Interleukin-18 binding proteins, their preparation and use for the treatment of sepsis

ABSTRACT

The present invention provides a method of treatment or prevention of sepsis and other diseases characteristic to the Systemic Inflammatory Response Syndrome (SIRS), including severe sepsis, septic shock, and sepsis related to cardiac dysfunction.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 11/505,554, filedAug. 16, 2006; which is a continuation-in-part application of U.S. Ser.No. 09/790,338, filed Feb. 21, 2001 (now U.S. Pat. No. 7,220,217); whichis a continuation-part application of U.S. Ser. No. 09/485,632, filedOct. 12, 2000 (now U.S. Pat. No. 6,605,280); and claims the benefit ofEP application 00103590.6, filed Feb. 21, 2000; EP application00103597.1, filed Feb. 21, 2000; EP application 00125633.8, filed Nov.23, 2000; PCT Application IL98/00379, filed Aug. 13, 1998; Israelapplication 125463, filed Jul. 22, 1998, Israel application 122134,filed Nov. 6, 1997; Israel application 121860, filed Sep. 29, 1997;Israel application 121639, filed Aug. 27, 1997; and Israel application121554, filed Aug. 14, 1997. Each of these applications is incorporatedby reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to interleukin-18 (IL-18) bindingproteins, hereinafter IL-18BP, capable of blocking the activity ofIL-18. This invention relates to soluble IL-18BPs obtainable from bodyfluids, to soluble IL-18BPs obtainable by expression of suitable DNAvectors in host cells, to virus-encoded homologues of IL-18BP obtainableby expression of suitable DNA vectors in host cells, to vectorsexpressing the various IL-18BPs, to vectors useful for expression ofIL-18BPs in humans and other mammals, to antibodies against IL-18BPs, totherapeutic use of said IL-18BPs in blocking IL-18 activity, totherapeutic use of said expression vectors in blocking IL-18 activityand to use of the antibodies.

More specifically, the present invention provides a method of treatmentand prevention of sepsis and other diseases characteristic to theSystemic Inflammatory Response Syndrome (SIRS), including severe sepsis,septic shock, and sepsis related to cardiac dysfunction.

BACKGROUND OF THE INVENTION

In 1989, an endotoxin-induced serum activity that induced interferon-γ(IFN-γ) obtained from mouse spleen cells was described (Micallef et al.,1996). This serum activity functioned not as a direct inducer of IFN-γbut rather as a co-stimulant together with IL-2 or mitogens. An attemptto purify the activity from post-endotoxin mouse serum revealed anapparently homogeneous 50-55 kDa protein. Since other cytokines can actas co-stimulants for IFN-γ production, the failure of neutralizingantibodies to IL-1, IL-4, IL-5, IL-6, or TNF to neutralize the serumactivity suggested it was a distinct factor. In 1995, the samescientists demonstrated that the endotoxin-induced co-stimulant forIFN-γ production was present in extracts of livers from micepreconditioned with P. acnes (Novick et al., 1992). In this model, thehepatic macrophage population (Kupffer cells) expand and in these mice,a low dose of bacterial lipopolysaccharide (LPS), which innon-preconditioned mice is not lethal, becomes lethal. The factor, namedIFN-γ-inducing factor (IGIF) and later designated interleukin-18(IL-18), was purified to homogeneity from 1,200 grams of P.acnes-treated mouse livers. Degenerate oligonucleotides derived fromamino acid sequences of purified IL-18 were used to clone a murine IL-18cDNA (Novick et al., 1992). IL-18 is an 18-19 kDa protein of 157 aminoacids, which has no obvious similarities to any peptide in thedatabases. Messenger RNAs for IL-18 and interleukin-12 (IL-12) arereadily detected in Kupffer cells and activated macrophages. RecombinantIL-18 induces IFN-gamma more potently than does IL-12, apparentlythrough a separate pathway (Novick et al., 1992). Similar to theendotoxin-induced serum activity, IL-18 does not induce IFN-γ by itself,but functions primarily as a co-stimulant with mitogens or IL-2. IL-18enhances T cell proliferation, apparently through an IL-2-dependentpathway, and enhances Th1 cytokine production in vitro and exhibitssynergism when combined with IL-12 in terms of enhanced IFN-γ production(Maliszewski et al., 1990).

Neutralizing antibodies to mouse IL-18 were shown to prevent thelethality of low-dose LPS in P. acnes pre-conditioned mice. Others hadreported the importance of IFN-γ as a mediator of LPS lethality inpre-conditioned mice. For example, neutralizing anti-IFN-γ antibodiesprotected mice against Shwartzman-like shock (Fantuzzi et al., 1998),and galactosamine-treated mice deficient in the IFN-γ receptor wereresistant to LPS-induced death (Byrn, 1990). Hence, it was notunexpected that neutralizing antibodies to murine IL-18 protected P.acnes-preconditioned mice against lethal LPS (Novick et al., 1992).Anti-murine IL-18 treatment also protected surviving mice against severehepatic cytotoxicity.

After the murine form was cloned, the human cDNA sequence for IL-18 wasreported in 1996 (Okamura et al., 1995). Recombinant human IL-18exhibits natural IL-18 activity (Okamura et al., 1995). Humanrecombinant IL-18 is without direct IFN-γ-inducing activity on humanT-cells, but acts as a co-stimulant for production of IFN-γ and otherT-helper cell-1 (Th1) cytokines (Okamura et al., 1995). To date, IL-18is thought of primarily as a co-stimulant for Th1 cytokine production(IFN-γ, IL-2 and granulocyte-macrophage colony stimulating factor)(Izaki, 1978) and also as a co-stimulant for FAS ligand-mediatedcytotoxicity of murine natural killer cell clones (Novick et al., 1989).

By cloning IL-18 from affected tissues and studying IL-18 geneexpression, a close association of this cytokine with an autoimmunedisease was found. The non-obese diabetic (NOD) mouse spontaneouslydevelops autoimmune insulitis and diabetes, which can be accelerated andsynchronized by a single injection of cyclophosphamide. IL-18 mRNA wasdemonstrated by reverse transcriptase PCR in NOD mouse pancreas duringearly stages of insulitis. Levels of IL-18 mRNA increased rapidly aftercyclophosphamide treatment and preceded a rise in IFN-γ mRNA, andsubsequently diabetes. Interestingly, these kinetics mimic that ofIL-12-p40 mRNA, resulting in a close correlation of individual mRNAlevels. Cloning of the IL-18 cDNA from pancreas RNA followed bysequencing revealed identity with the IL-18 sequence cloned from Kupffercells and in vivo pre-activated macrophages. Also NOD mouse macrophagesresponded to cyclophosphamide with IL-18 gene expression whilemacrophages from Balb/c mice treated in parallel did not. Therefore,IL-18 expression is abnormally regulated in autoimmune NOD mice andclosely associated with diabetes development (Novick et al., 1992).

IL-18 plays a potential role in immunoregulation or in inflammation byaugmenting the functional activity of Fas ligand on Th1 cells (Conti etal., 1997). IL-18 is also expressed in the adrenal cortex and thereforemight be a secreted neuro-immunomodulator, playing an important role inorchestrating the immune system following a stressful experience(Chater, 1986).

In vivo, IL-18 is formed by cleavage of pro-IL-18, and its endogenousactivity appears to account for IFN-γ production in P. acnes andLPS-mediated lethality. Because of its activity, blocking the biologicalactivity of IL-18 in human disease is a therapeutic strategy in manydiseases. This can be accomplished using soluble receptors or blockingantibodies to the cell-bound IL-18 receptor.

Cytokine binding proteins (soluble cytokine receptors) correspond to theextracellular ligand binding domains of their respective cell surfacecytokine receptors. They are derived either by alternative splicing of apre-mRNA, common to the cell surface receptor, or by proteolyticcleavage of the cell surface receptor. Such soluble receptors have beendescribed in the past, including among others, the soluble receptors ofIL-6 and IFN-γ (Nakamura et al., 1989), TNF (Dao et al., 1996; Engelmannet al., 1989), IL-1 and IL-4 (John, 1986), IFN-α/β (Mizushima andNagata, 1990) and others. One cytokine-binding protein, namedosteoprotegerin (OPG, also known as osteoclast inhibitory factor—OCIF),a member of the TNFR/Fas family, appears to be the first example of asoluble receptor that exists only as a secreted protein (Anderson, 1997;Bolton, 1980). The present invention deals with soluble IL-18 bindingproteins.

Recently, it has been suggested that the interleukin IL-18 is involvedin the progression of pathogenicity in chronic inflammatory diseases,including endotoxin shock, hepatitis, and autoimmune-diabetes(Kahiwamura and Okamura, 1998). A further indication of a possible roleof IL-18 in the development of liver injury resulted from experimentspublished by Tsuij et al. (Tsuij et al., 1999), showing an elevatedlevel of IL-18 in lipopolysaccharide-induced acute liver injury in amouse model. However, the mechanism of the multi-functional factor IL-18in the development of liver injury has not been elucidated so far.

Liver damage or injury may have diverse causes. It may be due to viralor bacterial infections, alcohol abuse, immunological disorders, orcancer, for example.

Viral hepatitis, due to Hepatitis B virus and Hepatitis C virus, forexample, are poorly managed diseases that afflict large number of peopleworldwide. The number of known hepatitis viruses known is constantlyincreasing. Apart from Hepatitis B and C virus, at least four otherviruses causing virus-associated hepatitis have been discovered so far,called Hepatitis A, D, E and G-Virus.

Alcoholic liver disease is another widespread disease associated withchronic consumption of alcohol. Immune hepatitis is a rare autoimmunedisease that is poorly managed. Liver injury also includes damages ofthe bile ducts. Primary biliary cirrhosis (PBC) is an autoimmune liverdisease characterized by destruction of the intrahepatic bile ducts.

Several studies have demonstrated that damage to the liver in diseasessuch as alcoholic hepatitis, liver cirrhosis, viral hepatitis andprimary biliary cirrhosis is associated with T-helper cell-1 (Th1)responses. In one study, a novel liver injury model was established inmice by targeting of ovalbumin-containing liposomes into the liver,followed by adoptive transfer of ovalbumin-specific Th1 cells. Combinedtreatment of mice with ovalbumin-containing liposomes and Th1 celltransfer caused an increase in serum transaminase activity that wasparalleled with an elevation of serum IFN-γ levels. In sharp contrast,ovalbumin-specific Th2 cell transfer resulted in an increase of serumIL-4 levels but did not induce liver injury. The liver injury wasblocked by anti-IFN-γ antibodies and anti-tumor necrosis factor (TNF)-αantibodies. These findings indicate that Th1 cells are the majoreffector cells in acute liver injury (Nishimura and Ohta, 1999) Inanother set of studies it was shown that mice over-expressing IFN-γexhibit spontaneous hepatitis without any pathogen or any otherstimulant (Okamoto et al., 1998).

Another study implicated Th1 responses in primary biliary cirrhosis(PBC). PBC is an autoimmune liver disease characterized by destructionof the intrahepatic bile ducts. It is generally believed that cellularimmune mechanisms, particularly involving T cells, result in this bileduct damage. The relative strength of Th1 and Th2 responses has recentlybeen proposed to be an important factor in the pathophysiology ofvarious autoimmune diseases. In this study, the The subset balance inPBC was evaluated by detection of cytokines specific to the two T-cellsubsets, i.e., IFN-γ for Th1 cells and IL-4 for Th2 cells. IFN-γ andIL-4 messenger RNA (mRNA) positive cells were counted in liver sectionsfrom 18 patients with PBC and 35 disease controls including chronicactive hepatitis C, extrahepatic biliary obstruction, and normal liver,using nonisotopic in situ hybridization and immunohistochemistry.Mononuclear cells expressing IFN-γ and IL-4 mRNA were aggregated ininflamed portal tracts in PBC livers, but were rarely present inextrahepatic biliary obstruction, alcoholic fibrosis, or normal liversections. The IFN-γ and IL-4 mRNA positive cells in PBC livers weredetected in significantly higher numbers than in control livers(P<0.01). Moreover, IFN-γ mRNA expression was more commonly detectedthan IL-4 expression in PBC livers, and the levels of IFN-γ mRNAexpression were highly correlated with the degree of portal inflammatoryactivity. IFN-γ mRNA-positive cells were detected primarily arounddamaged bile ducts that were surrounded by lymphoid aggregates. The dataindicate that Th1 cells are the more prominent T-cell subset in thelymphoid infiltrates in PBC (Harada et al., 1997).

The cytokine pattern on viral antigen recognition is also believed toexert a profound influence on the resolution of viral infections andviral clearance. One study investigated whether a cytokine imbalanceoriented toward Th2 type response plays a role in chronic hepatitis B.Cytokine profiles of peripheral blood mononuclear cells associated withchronic hepatitis B were analyzed by RT-PCR. Upon hepatitis B surfaceantigen (HbsAg) stimulation, expression of IFN-γ, IL-2, IL-4, and IL-10was detected in 41%, 8%, 41%, and 50% of the patients, respectively.Among these cytokines, the expression of the Th1 cytokine IFN-γ wasassociated with high levels of serum AST/ALT (Aspartateaminotransferase/Alanine aminotransferase), representing typical markersof liver damage. Th2 type cytokines were not shown to exert a protectiveeffect on hepatocytes. In conclusion, production of a Th1 cytokine,IFN-γ, by HBsAg-reactive cells was associated with hepatocyte damage inchronic hepatitis B (Lee et al., 1999). High levels of the FAS ligandand its receptor (CD95) were reported in liver of hepatitis B patients(Luo et al., 1997). FAS ligand is considered to be one of the majorcytotoxic agents leading to hepatocyte apoptosis.

Another study identified factors associated with the progression ofliver injury in 30 hepatitis C virus/RNA (HCV/RNA)-positive untreatedpatients with chronic hepatitis. Necroinflammatory and architecturaldamage were evaluated using Ishak's score. Activated hepatic stellatecells (HSC) were visualized by immunohistochemistry for α-smooth muscleactin (αSMA) and quantitated by morphometry. Plasma HCV/RNA wasevaluated using a competitive RT-PCR method. To study the type of immuneresponse involved in the progression of liver injury, IFN-γ-positivecells (as expression of a Th1-like response) were evaluated byimmunohistochemistry and quantitated by morphometry. It was found thatHSC were mostly detected close to areas of lobular necroinflammation orlining fibrotic septa. The αSMA- and Sirius Red-positive parenchymacorrelated significantly with necroinflammatory and architecturalscores. IFNγ-positive cells were detected in periportal areas associatedwith the inflammatory infiltrates and significantly correlated witharchitectural damage. It was therefore concluded that HSC activation andprogression of liver injury are associated with a Th1-like response(Baroni et al, 1999). Similarly to the case of Hepatitis B, FAS ligandand its receptor were found in liver and sera of hepatitis C patients(Hiramatsu et al, 1994; Okazaki et al, 1996; Lio et al., 1998)

Th1 cytokines and other Th1 markers were found to be associated withalcoholic hepatitis and liver cirrhosis. Inflammatory stimuli and lipidperoxidation activate nuclear factor κ B (NF-κB) and upregulateproinflammatory cytokines and chemokines. In one study, the relationshipbetween pathological liver injury, endotoxemia, lipid peroxidation, andNF-κB activation and imbalance between pro- and anti-inflammatorycytokines was evaluated. Rats (5 per group) were fed ethanol and a dietcontaining saturated fat, palm oil, corn oil, or fish oil byintragastric infusion. Dextrose isocalorically replaced ethanol incontrol rats. Pathological analysis was performed and measurements ofendotoxin were taken, lipid peroxidation, NF-κB, and messenger RNA(mRNA) levels of proinflammatory cytokines (TNFα, IL-1 beta, IFN-γ, andIL-12), C—C chemokines (regulated upon activation, normal T cellexpressed and secreted [RANTES], monocyte chemotactic protein [MCP]-1,macrophage inflammatory protein [MIP]-1-α), C—X—C chemokines (cytokineinduced neutrophil chemoattractant [CINC], MIP-2, IP-10, and epithelialneutrophil activating protein [ENA]-78), and anti-inflammatory cytokines(IL-10, IL-4, and IL-13). Activation of NF-κB and increased expressionof proinflammatory cytokines C—C and C—X—C chemokines was seen in therats exhibiting necroinflammatory injury (fish oil-ethanol and cornoil-ethanol). These groups also had the highest levels of endotoxin andlipid peroxidation. Levels of IL-10 and IL-4 mRNA were lower in thegroup exhibiting inflammatory liver injury. Thus, activation of NF-κBoccurs in the presence of proinflammatory stimuli and results inincreased expression of Th1 proinflammatory cytokines and chemokines(Naji et al., 1999). FAS ligand and its receptor are also elevated inalcoholic liver diseases, suggesting once again that Th1 cytokines areinvolved in the autoimmune processes induced in alcoholic hepatitis(Galle et al., 1995; Taieb et al, 1998; Fiore et al., 1999).

TNF-α has also emerged as a common pathway in the pathogenesis ofalcohol-related hepatic necro-inflammation. Increased levels of hepaticand serum TNF have been documented in animal models of alcoholic liverdisease and in human alcoholic liver disease. This dysregulated TNFmetabolism has been postulated to play a role in many of the metaboliccomplications and the liver injury of alcoholic liver disease (Grove etal., 1997; McClain and Cohen, 1989). For instance it was found in onestudy that patients with alcoholic hepatitis had higher TNF-α levels(mean, 26.3 ng/L; 95% Cl, 21.7 to 30.9) than normal subjects (6.4 ng/L;Cl, 5.4 to 7.4). Patients who subsequently died had a higher TNF-α level(34.7 ng/L; Cl, 27.8 to 41.6) than survivors (16.6 ng/L; Cl, 14.0 to19.2). In patients with alcoholic hepatitis, TNF-α levels correlatedpositively with serum bilirubin (r=0.74; P=0.0009) and serum creatinine(r=0.81; P=0.0003). Patients with alcoholic hepatitis had higher TNF-αlevels than patients with inactive alcoholic cirrhosis (11.1 ng/L; Cl,8.9 to 13.3) and severely alcoholic persons without liver disease (6.4ng/L; Cl, 5.0 to 7.8). Patients with abnormal renal function had lowerTNF-α levels (14.1 ng/L; Cl, 5.4 to 22.8) than patients with alcoholichepatitis. It was therefore concluded that elevations in TNF-α inalcoholic hepatitis are most marked in severe cases, suggesting thatTNF-α plays a role in the pathogenesis (Bird et al., 1990)

TNF mediates many of the biologic actions of endotoxin. Recent studieshave shown that TNF administration may cause liver injury and that TNFmay mediate the lethality of the hepatotoxin galactosamine. One of themost potent TNF inducers is endotoxin. Because patients with alcoholicliver disease frequently have endotoxemia and because many of theclinical manifestations of alcoholic hepatitis are known biologicactions of TNF, its activity was evaluated in patients with alcoholichepatitis. Basal and lipopolysaccharide-stimulated TNF release fromperipheral blood monocytes, a major source of TNF production, wasdetermined in 16 patients with alcoholic hepatitis and 16 healthyvolunteers. Eight of 16 alcoholic hepatitis patients and only two of 16healthy volunteers had detectable spontaneous TNF activity (p less than0.05). After lipopolysaccharide stimulation, mean monocyte TNF releasefrom alcoholic hepatitis patients was significantly increased to overtwice that of healthy controls (25.3+/−3.7 vs. 10.9+/−2.4 units per ml,p less than 0.005). It was therefore concluded that monocytes fromalcoholic hepatitis patients have significantly increased spontaneousand lipopolysaccharide-stimulated TNF release compared to monocytes fromhealthy volunteers (McClain and Cohen, 1989.

Lipopolysaccharide (LPS)-binding protein (LBP) and CD14 play keyintermediary roles in the activation of cells by endotoxin. Gut-derivedLPS has been postulated to participate in promoting pathological liverinjury in alcoholic liver disease. It was demonstrated that rats fedintragastrically with ethanol in oil for 4 weeks had elevated levels ofCD14 and LBP in their Kupffer cells and hepatocytes, respectively.Expression of CD14 mRNA was also elevated in nonmyeloid cells. EnhancedLBP and CD14 expression rapidly increases the LPS-induced expression ofvarious pro-inflammatory cytokines and correlates with the presence ofpathological liver injury in alcoholic liver injury (Su et al., 1998;Lukkari et al., 1999).

IL-6 is a 26 kd cytokine that plays a major role in the acute phaseresponse, especially the hepatic aspects of the acute phase response.Patients with alcoholic hepatitis manifest many aspects of the acutephase response. Serial plasma IL-6 levels in 30 consecutive patientswith moderate to severe alcoholic hepatitis was measured during 6 month.Mean admission plasma IL-6 activity was markedly increased (49.8+/−8.5U/ml, normal less than 5 U/ml) in patients with alcoholic hepatitis, andlevels decreased with clinical improvement to 15.6+/−6.1 U/ml at 6months. Admission IL-6 activity correlated significantly (r=0.82) withthe severity of liver disease as assessed by the discriminant functionof Maddrey. IL-6 activity fell over time in a pattern similar to that ofbilirubin and C-reactive protein. This and additional studies suggestthat plasma IL-6 is probably a marker of inflammation and severity ofdisease in alcoholic hepatitis (Sheron et al., 1991; Hill et al., 1992).

IL-8, a cytokine produced by a number of cells, including monocytes,macrophages, Kupffer cells and hepatocytes, can activate neutrophils.Peripheral neutrophilia and liver neutrophil infiltration are frequentlynoted in patients with alcoholic liver disease. It was found that serumIL-8 levels were markedly elevated in patients with alcoholic hepatitis(437+/−51 pg/ml) when compared to all other groups (p<0.05). Levels ofIL-8 in patients with alcoholic fatty liver, alcoholic cirrhosis andviral hepatitis were higher than those in controls and in patients withnon-alcoholic fatty liver. In addition, IL-8 levels were higher inpatients who died (p=0.007), and correlated with biochemical andhistological parameters, and severity of liver injury: serum aspartateaminotransferase, alanine aminotransferase, total bilirubin, prothrombintime, indocyanine green retention ratio, TNF-α and peripheral neutrophilcount in patients with alcoholic hepatitis. After a 2-year follow up,patients with IL-8 above 479 pg/ml had a higher mortality rate in thealcoholic hepatitis group (p=0.033). These findings suggest that IL-8 aswell as some other chemokines are activated in alcoholic liver disease,especially in alcoholic hepatitis, and is closely correlated with liverinjury (Martinez et al., 1992; Hill et al., 1992).

Induction of adhesion molecules such as ICAM-1 is associated with theactivation and attraction of a special population of inflammatory cellscharacteristic for alcoholic hepatitis. Frozen liver samples frompatients who died with signs of acute alcoholic hepatitis show elevatedICAM-1 expression in the membranes of hepatocytes, as well as theoccurrence of CD11b positive polymorphonuclear leukocytes (neutrophils)suggesting a possible major role of the beta 2-integrin Mac-1 as aligand for ICAM-1. It was concluded that in alcoholic hepatitiscytokines may be responsible for the induction of the adhesion moleculeICAM-1 on hepatocytic membranes and activate a defined population ofinflammatory cells, thus contributing to the characteristic histologicalpicture of acute alcoholic hepatitis with its concentration ofneutrophils especially in areas with ballooned Mallory body-containinghepatocytes (Afford et al., 1998).

A significant increase in both NK cells (CD3−/CD56+) and the cytotoxic Tcells coexpressing the CD3 and the CD56 molecules together with anincreased NK cytotoxic activity were observed in patients havingalcoholic hepatitis. Interestingly these abnormalities persisted duringthe withdrawal period (Ohlinger et al., 1993).

Arthritis is a disease involving joint inflammation. The joints showswelling, stiffness, tenderness, redness or warmth. The symptoms may beaccompanied by weight loss, fever or weakness. When these symptoms lastfor more than two weeks, inflammatory arthritis e.g. rheumatoidarthritis may be the cause. Joint inflammation may also be caused byinfection, which can lead to septic arthritis. A very common type ofarthritis is degenerative joint disease (osteoarthritis). Jointinflammation is not a prominent feature of osteoarthritis.

The medicaments commonly prescribed for arthritis and related conditionsare non-steroidal anti-inflammatory drugs (NSAIDs). NSAIDs includeaspirin and aspirin-like drugs. They reduce inflammation, which is thecause for joint pain, stiffness and swelling of the joints. However,NSAIDs are unspecific drugs having a number of side effects, involvingbleeding of the stomach (Homepage of the Department of Orthopaedics ofthe University of Washington on Arthritis, Frederick Matsen (Chairman)).In addition to NSAIDs, Celebrex™, a cyclooxygenase (COX-2) inhibitor, isused to relieve the signs and symptoms of osteoarthritis and rheumatoidarthritis in adults. It is also indicated for the treatment of patientswith familial adenomatous polyposis.

Further, TNF antagonists are used for the treatment of arthritis. TNFantagonists are described, for example, in WO 9103553.

Recent studies indicate that the interleukin IL-18 plays aproinflammatory role in joint metabolism. Olee et al. (1999) showed thatIL-18 is produced by articular chondrocytes and induces proinflammatoryand catabolic responses. The IL-18 mRNA was induced by IL-18 inchondrocytes. Chondrocytes produced the IL-18 precursor and in responseto IL-1 stimulation secreted the mature form of IL-18. Studies on IL-18effects on chondrocytes further showed that it inhibits TGF-β-inducedproliferation and enhances nitric oxide production. IL-18 stimulated theexpression of several genes in normal human articular chondrocytesincluding inducible nitric oxide synthase, inducible cyclooxygenase,IL-6, and stromelysin. Gene expression was associated with the synthesisof the corresponding proteins. Treatment of normal human articularcartilage with IL-18 increased the release of glycosaminoglycans. Thesefinding identified IL-18 as a cytokine that regulates chondrocyteresponses and contributes to cartilage degradation.

The localisation of Interleukin-1β-converting enzyme (ICE)/caspase-1 inhuman osteoarthritic tissues and its role in the maturation ofinterleukin-1 beta and interleukin-18 have been shown by Saha et al.(1999). Saha et al. studied the expression and production of caspase-1in human normal and osteoarthritic (OA) cartilage and synovium,quantitated the level of ICE in OA chondrocytes, and examined therelationship between the topographic distribution of ICE, interleukin-1β(IL-1β), and IL-18, as well as apoptosis of chondrocytes. Theexperiments performed in this study indicated that ICE was expressed andsynthesised in both human synovial membrane and cartilage, with asignificantly greater number of cells staining positive in OA tissuethan in normal tissue. ICE production was preferentially located in thesuperficial and upper intermediate layers of articular cartilage. Theproduction of mature IL-1beta in OA cartilage explants and chondrocyteswas completely blocked by treatment with a specific ICE inhibitor, whichalso markedly diminished the number of IL-18-positive cells. Therelationship between active IL-1beta and ICE suggests that ICE maypromote OA progression by activating this proinflammatory cytokine, andthat IL-18 may play a role in cartilage pathology.

Gracie et al. (1999) suggested a proinflammatory role for IL-18 inrheumatoid arthritis. Gracie et al. detected the IL-18 mRNA and proteinwithin rheumatoid arthritis synovial tissues in significantly higherlevels than in osteoarthritis controls. It was also shown that acombination of IL-12 or IL-15 with IL-18 induced the IFN-γ production bysynovial tissues in vitro. Furthermore, IL-18 administration ofcollagen/inclomplete Freund's adjuvant-immunized mice facilitated thedevelopment of an erosive, inflammatory arthritis, suggesting that IL-18may be proinflammatory in vivo.

However, so far, apart from chemical compounds, only the blockade ofTNFα and IL-18 by using soluble receptors or monoclonal antibodies havebeen shown to decrease murine collagen-induced arthritis (CIA, which isa mouse model for rheumatoid arthritis) (Williams et al., 1994), andwere therefore suggested as a therapeutic for rheumatoid arthritis.

The term “systemic inflammatory response syndrome (SIRS)” describes thefamiliar clinical syndrome of sepsis, independent of its cause. SIRS canresult from trauma, pancreatitis, drug reactions, autoimmune disease,and other disorders; when it arises in response to infection, sepsis issaid to be present (Nathens and Marshall 1996).

Septic shock is the most common cause of death in medical and surgicalintensive-care units (Aestiz and Rackow 1998). The terms sepsis, severesepsis and septic shock are used to identify the continuum of theclinical response to infection. Patients with sepsis present evidencesof infection and clinical manifestations of inflammation. Patients withsevere sepsis develop hypoperfusion with organ dysfunction. Septic shockis manifested by hypoperfusion and persistent hypotension. Mortalityranges from 16% in patients with sepsis to 40-60% in patients withseptic shock. Bacterial infection is the most common cause of septicshock. The most frequent sites of infection are the lungs, abdomen, andurinary tract. General anti-inflammatory therapies such as the use ofcorticosteroids, failed to show improvement in survival from sepsis andseptic shock. Three monoclonal antibodies specific to endotoxin havebeen tested in clinical trials and have failed to improve survivalrates. Therapy with antagonists to tumor necrosis factor, interleukin 1,bradykinin, ibuprofen, and platelet activating-factor, did not showimprovement on survival from septic shock (Aestiz and Rackow 1998).

Sepsis is caused inter alia by Gram-positive bacteria e.g.,Staphylococcus epidermidis. Lipoteichoic acids and peptidoglycans, whichare the main cell wall components of the Staphylococcus species, arethought to be the inducers of cytokine release in this condition (Gruptaet al. 1996 and Cleveland et al. 1996). However, other Gram-positivecomponents are considered as stimulators of cytokine synthesis as well(Henderson et al. 1996). Production of IL-1, TNF-α and IFN-γ are thoughtto be the major contributors in the pathogenesis of septic shock(Dinarello 1996 and Okusawa et al. 1988). In addition, the chemokineIL-8 was shown to be induced by neutrophils in response to S.epidermidis (Hachicha et al. 1998). Important factors in the regulationof IL-1, TNF-α and IFN-γ induction by S epidermidis are IL-18, IL-12,IL-1 β and TNF-α. IL-1β and TNF-α can be considered as a co-stimuli forIFN-γ production by T lymphocytes in a manner similar to IL-18. Thesetwo cytokines have a co-stimulatory activity on IFN-γ production in thecontext of IL-12 or bacterial stimulation (Skeen et al. 1995 and Trippat al. 1993).

Recently it was reported by Nakamura et al. (2000) that concomitantadministration of IL-18 and IL-12 to mice results in high toxicitysimilar to that found in endotoxin-induced septic shock.

The levels of IL-18 have been shown to be elevated in sera from patientswith sepsis (Endo et al. 2000) but this increase correlated withcreatinine levels suggesting that the elevated levels of IL-18 mayresult from renal failure. In another study (Grobmyer et al. 2000), thelevels of IL-18 in 9 subjects with sepsis during the first 96 hoursfollowing hospital admission were high and did not correlate withcreatinine levels.

Dayer (1999) summarized the different and partially contradictingfunctions of IL-18. IL-18 is a pleiotropic interleukin having bothinflammatory enhancing and attenuating functions. On the one hand, itenhances production of the proinflammatory cytokines like TNFα,therefore promoting inflammation. On the other hand, it induces theproduction of NO, an inhibitor of caspase-1, thus blocking thematuration of IL-1β and IL-18, and possibly attenuating inflammation.This ambiguous role of IL-18 seriously questioned the efficacy of IL-18inhibitors in inflammatory diseases. Furthermore, because of theinteraction of a huge variety of different cytokines and chemokines inthe regulation of inflammation, it could not have been expected toobtain a beneficial effect by blocking only one of the players in such acomplicated scenario.

Netea et al. (2000) reported that administration of anti-IL-18polyclonal antibody protected mice against the deleterious effects ofboth LPS derived from E. coli or S. typhimurium species tested,supporting the concept that IL-18 has an important pathogenic role inlethal endotoxemia. However, the uncertainty regarding the efficiency ofa potential treatment formulation based on administration of IL-18inhibitors, is manifested in that IL-18 knock out mice are not lesssusceptible to sepsis as compared to wild type animals (Sakao et al.1999) and in that recent reports suggest that the proinflammatoryactivity of IL-18 is essential to host defences against severeinfections (Nakanishi et al. 2001 and Foss et al. 2001).

Thus, there exists a need to provide means to treat and/or preventsepsis despite the above-mentioned considerations regarding the use ofIL-18 inhibitors.

SUMMARY OF THE INVENTION

The present invention provides IL-18 binding proteins (IL-18BPs) andvirally encoded IL-18BP homologues (hereinafter, viral IL-18BPs), andfused proteins, muteins, functional derivatives and active fragmentsthereof, capable of binding to IL-18. The invention also provides aprocess for obtaining IL-18BPs by isolating them from human fluids toobtain IL-18BP, or by recombinant means, which also allows forattainment of the various IL-18BPs. The invention also providesexpression vectors of IL-18BPs, suitable for expression of IL-18BP inhumans and other mammals. The IL-18BPs and the expression vectors of thepresent invention are useful for blocking the biological activities ofIL-18.

Replicable expression vehicles containing DNAs suitable for expressionof the various IL-18BPs in host cells, host cells transformed herewithand proteins and polypeptides produced by expression of such hosts arealso provided.

The invention further provides antibodies to the IL-18BPs and the viralIL-18BPs, suitable for affinity purification and immunoassays of same.

The invention further provides pharmaceutical compositions consisting ofsuitable vehicles and IL-18BPs, or viral IL-18BPs, or vectors forexpressing same in humans and other mammals, for the treatment ofdiseases or conditions which require modulation of IL-18 activity.

These diseases or conditions include autoimmune diseases, type Idiabetes, rheumatoid arthritis, graft rejections, inflammatory boweldisease, sepsis, multiple sclerosis, ischemic heart diseases (includingheart attacks), ischemic brain injury, chronic hepatitis, psoriasis,chronic pancreatitis, acute pancreatitis and the like.

It is a specific object of the present invention to provide for a novelmeans for treating and/or preventing liver injury. It has been foundthat an IL-18 inhibitor is effective in the prevention and treatment ofliver damages. The invention therefore also relates to the use of anIL-18 inhibitor for the manufacture of a medicament for treatment and/orprevention of liver injury. More specifically, the invention relates tothe treatment and/or prevention of liver injuries caused by alcoholichepatitis, viral hepatitis, immune hepatitis, fulminant hepatitis, livercirrhosis, and primary biliary cirrhosis.

It has also been found in accordance with the present invention that aninhibitor of IL-18 is effective in the therapy of arthritis. Thetherapeutic effect includes decreasing the severity of the disease, aswell as preventing the spreading of the disease. The invention thereforerelates to the use of an inhibitor of IL-18 for treatment and/orprevention of arthritis. This finding is unexpected, since from thestate of the art outlined above, it could not have been concluded that ablockade of one specific factor involved in arthritis, namelyinterleukin IL-18, would lead to the alleviation of arthritis or eventhe curing of a diseased arthritic joint.

It has also been found that the administration of an IL-18 inhibitorsignificantly diminishes cartilage erosion in a murine model ofarthritis. The present invention thus also relates to the use of aninhibitor of IL-18 in the manufacture of a medicament for treatmentand/or prevention of cartilage destruction.

In order to apply a gene therapeutic approach to deliver the IL-18inhibitor to the diseased tissue or cell, it is a further object of theinvention to use an expression vector comprising the coding sequence ofan IL-18 inhibitor for the treatment and/or prevention of arthritis.

The invention relates to the use of an inhibitor of IL-18 in themanufacture of a medicament for the treatment and/or prevention ofsepsis and other diseases characteristic to the Systemic InflammatoryResponse Syndrome (SIRS) selected from severe sepsis and septic shock,and also for sepsis related cardiac dysfunction.

More specifically the invention relates to the use of an inhibitor ofIL-18, selected from caspase-1 (ICE) inhibitors, antibodies againstIL-18, antibodies against any of the IL-18 receptor subunits, inhibitorsof the IL-18 signaling pathway, antagonists of IL-18 which compete withIL-18 and block the IL-18 receptor, inhibitors of IL-18 production andIL-18 binding proteins, isoforms, muteins, fused proteins, functionalderivatives, active fractions or circularly permutated derivativesthereof having at least essentially the same activity as an IL-18binding protein.

The IL-18 binding protein used according to the invention is an isoform,a mutein, fused protein (e.g., Ig fused), functional derivative (e.g.,PEG-conjugated), active fraction or circularly permutated derivativethereof.

The antibodies used according to the invention may be anti IL-18specific antibodies selected from chimeric, humanized and humanantibodies.

In addition, the invention relates to the use of an inhibitor in themanufacture of a medicament further comprising an IL-12 inhibitor,preferably an IL-12 neutralizing antibody, an interferon, preferablyinterferon .alpha. or .beta., a tumor necrosis factor inhibitor,preferably soluble TNFRI or TNFRII, and/or an IL-1 inhibitor, preferablyIL-1 receptor antagonist for simultaneous, sequential or separateadministration.

In one aspect, the invention provides the use of an expression vectorcomprising the coding sequence of an inhibitor of IL-18 selected fromcaspase-1 (ICE) inhibitors, antibodies against IL-18, antibodies againstany of the IL-18 receptor subunits, inhibitors of the IL-18 signalingpathway, antagonists of IL-18 which compete with IL-18 and block theIL-18 receptor, inhibitors of IL-18 production and IL-18 bindingproteins, isoforms, muteins, fused proteins, or circularly permutatedderivatives thereof having at least essentially the same activity as anIL-18 binding protein, in the manufacture of a medicament for thetreatment and/or prevention of sepsis, for example by gene therapy.

In another aspect, the invention provides the use of a vector forinducing and/or enhancing the endogenous production of an inhibitor ofIL-18 in a cell, in the manufacture of a medicament for the treatmentand/or prevention of sepsis.

In addition, the invention provides the use of a cell that has beengenetically modified to produce an inhibitor of IL-18 in the manufactureof a medicament for the treatment and/or prevention of sepsis.

In another embodiment, the invention relates to a method for thetreatment and/or prevention of sepsis and other diseases characteristicto the Systemic Inflammatory Response Syndrome (SIRS), including sepsisrelated cardiac dysfunction, comprising administrating to a subject inneed thereof a pharmaceutically effective amount of inhibitor of IL-18selected from caspase-1 (ICE) inhibitors, antibodies against IL-18,antibodies against any of the IL-18 receptor subunits, inhibitors of theIL-18 signaling pathway, antagonists of IL-18 which compete with IL-18and block the IL-18 receptor, inhibitors of IL-18 production, and IL-18binding proteins, isoforms, muteins, fused proteins, functionalderivatives, active fractions or circularly permutated derivativesthereof having essentially the same activity as an IL-18 bindingprotein. The method may comprise co-administration of a therapeuticallyeffective amount an inhibitor of cytokines selected from IL-12inhibitor, preferably neutralizing antibodies, Tumor Necrosis Factorinhibitors, preferably soluble portion of TNFRI or TNFRII), IL-1inhibitors. preferably the IL-1 receptor antagonists and IL-8inhibitors, and/or an interferon, preferably interferon-.alpha. or-.beta.

In addition, the invention provides a method for the treatment and/orprevention of sepsis and other diseases characteristic to the SystemicInflammatory Response Syndrome (SIRS) comprising administrating to asubject in need thereof a pharmaceutically effective amount of vectorcoding the sequence of an inhibitor of IL-18 selected from caspase-1(ICE) inhibitors, antibodies against IL-18, antibodies against any ofthe IL-18 receptor subunits, inhibitors of the IL-18 signaling pathway,antagonists of IL-18 which compete with IL-18 and block the IL-18receptor, inhibitors of IL-18 production, and IL-18 binding proteins,isoforms, muteins, fused proteins or circularly permutated derivativesthereof.

In a further embodiment, the invention relates to a method for thetreatment and/or prevention of sepsis and other diseases characteristicto the SIRS comprising administrating to a subject in need thereof apharmaceutically effective amount of a vector for inducing and/orenhancing the endogenous production of an inhibitor of IL-18 in a cell.

In addition, the invention provides a method for the treatment and/orprevention of sepsis and other diseases characteristic to the SIRScomprising administration to a subject in need thereof a cell that hasbeen genetically modified to produce an inhibitor of IL-18.

The invention also relates to the use of an inhibitor of IL-18 in themanufacture of a medicament for the treatment and/or prevention ofsepsis and other diseases characteristic to the Systemic InflammatoryResponse Syndrome (SIRS) selected from severe sepsis, septic shock, andalso for sepsis related to cardiac dysfuntion.

The invention includes a method of treating or preventing sepsisassociated with an excess of IL-18, where the method comprisesadministering to a subject in which such treatment or prevention isdesired, a composition comprising (a) a polypeptide comprising an aminoacid sequence selected from the group consisting of AA1-AA192 of SEQ IDNO:2, or AA29-AA192 of SEQ ID NO:2, (b) a nucleic acid moleculecomprising a nucleic acid sequence encoding a polypeptide comprising anamino acid sequence selected from the group consisting of AA1-AA192 ofSEQ ID NO:2, or AA29-AA192 of SEQ ID NO:2, or (c) a mutein of any one ofthe sequences in (a) or (b), characterized in that the mutein i) has atleast 90% identity to at least one of the sequences in (a); ii)comprises the amino acid sequence of SEQ ID NO:10; and iii) binds IL-18in an amount sufficient to treat or prevent the disease in the subject.

In one embodiment, the method of treating or preventing sepsis includesadministration to a human subject. In another embodiment, thepolypeptide is glycosylated at one or more sites. Alternatively, thepolypeptide is not glycosylated. In another embodiment, the polypeptidefurther comprises at least one moiety attached to one or more functionalgroups which occur as one or more side chains on the amino acid residuesor the N- or C-terminal groups.

In another aspect of the method of treating sepsis, the polypeptide iscircularly permutated. In another aspect, the polypeptide is a non-viralprotein. In another the polypeptide is a human protein. In anotheraspect, the polypeptide is a fused protein. In the polypeptide comprisesan Ig fusion. In another aspect, the polypeptide is soluble. In anotheraspect, the polypeptide is pegylated. In another aspect, at least onemoiety is a polyethylene glycol moiety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows SDS-PAGE (sodium dodecyl sulfate polyacrylamide gelelectrophoresis) of ligand affinity purified IL-18 binding protein.Crude urinary proteins (concentrated by ultrafiltration of 500 L normalhuman urine) were loaded on an IL-18-agarose column. The column waswashed and bound proteins eluted at pH 2.2. Eluted fractions wereneutralized and aliquots were analyzed by SDS-PAGE (10% acrylamide)under non-reducing conditions and silver staining. The lanes are: 1:crude urinary proteins (1.5 μg, loaded on the gel); 2-9: elutions 1-8,respectively, from the IL-18-agarose column; 10: molecular weightmarkers, in kD, as indicated on the right side. An arrow indicates theband corresponding to IL-18BP.

FIG. 2 shows an autoradiogram of SDS-PAGE (7.5% acrylamide) of complexesconsisting of ¹²⁵I-IL-18 (apparent molecular weight 19 kD), cross-linkedto the following preparations of soluble IL-18 binding protein: Lane 1:Wash of the IL-18 affinity column. Lane 2: Elution 2 of the IL-18affinity column. Lane 3: Elution 3 of the IL-18 affinity column.Molecular weight markers are indicated on the right side (in kD). Anarrow indicates the cross-linked product (58 kD).

FIG. 3 shows inhibition of IL-18-induced production of IFN-γ by IL-18BP

-   -   (A) Mouse splenocytes were stimulated (24 hr, 37° C.) with the        indicated combinations of LPS (1 μg/ml) and human IL-18 (5        ng/ml), added either directly, or after pre-mixing (1 h, 37° C.)        with urinary IL-18BP. The level of mulFN-γ in the culture was        determined after 24 hr.    -   (B) Mouse splenocytes were incubated (24 h) with LPS (1 μg/ml)        together with murine IL-18 (10 ng/ml) pre-mixed (1 h, 37° C.)        with increasing concentrations of human IL-18BP.    -   (C) Mouse splenocytes were incubated (24 h) with LPS (10 μg/ml)        together with increasing concentrations of human IL-18BP.    -   (D) Mouse splenocytes were incubated (24 h) with Con A (1        μg/ml), together with increasing concentrations of human        IL-18BP.    -   (E) Human peripheral blood mononuclear cells (PBMC) of three        donors were stimulated with IL-12 (10 ng/ml) and huIL-18 (25        ng/ml), added either alone, or after pre-mixing (1 h, 37° C.)        with urinary IL-18BP. Human KG-1 cells were stimulated with        TNF-α (20 ng/ml) and huIL-18 (25 ng/ml), added either alone, or        after pre-mixing (1 h, 37° C.) with urinary IL-18BP.

FIG. 4 shows the effect of recombinant IL-18BP on human and mouse IL-18activity. His₆-tagged IL-18BPa was transiently expressed in COS7 cellsand purified.

-   -   (A) Human IL-18 (5 ng/ml) was pre-mixed with either        His₆-tagged-IL-18BPa or RPMI and added to mouse spleen cells        together with LPS (1 μg/ml). IFN-γ production was measured after        24 h.    -   (B) Mouse IL-18 (10 ng/ml) was pre-mixed with either        His₆-tagged-IL-18BPa or RPMI and added to mouse spleen cells        together with LPS (1 μg/ml). IFN-γ production was measured after        24 h.    -   (C) Human IL-18 (25 ng/ml) was pre-mixed with either        COS7-IL-18BPa or RPMI and added to Human PBMC in the presence of        IL-12 (10 ng/ml).    -   (D) Human IL-18 (25 ng/ml) was pre-mixed with either        COS7-IL-18BPa or RPMI and added to Human KG-1 cells in the        presence of TNF-α (20 ng/ml).

FIG. 5 shows a histogram depicting the serum levels of IFN-γ (pg/ml)after injection of various amounts of recombinant IL18BP (0; 0.04; 0.4;4 mg/kg) into mice 1 h before the injection of LPS. Blood samples weretaken 5 h after LPS injection and analyzed by ELISA for circulatingIFN-γ.

FIG. 6 shows a histogram depicting the serum levels of Alanineaminotransferase. Mice were injected with increasing doses ofrecombinant human IL18BP (0; 0.04; 0.4; 4 mg/kg) before injection of LPSinto P. acnes sensitized mice. Blood samples were taken 5 h after LPSinjection and serum levels of Alanine aminotransferase (ALT) weremeasured. SF=Sigma-Frankel: 1 SF Unit of AST/ALT will form 4.82×10⁻⁴μmol glutamate/minute at pH 7.5 at 25° C.

FIG. 7 shows the survival time of the mice after LPS injection. Micewere injected with different doses of recombinant human IL18BP (0; 0.04;0.004; 4 mg/kg) 20 min before injection of LPS into P. acnes sensitizedmice. Triangles: 4 mg/kg; small diamond: 0.4; big diamond: 0.04;circles: no IL18BP (only LPS).

FIG. 8 shows a histogram depicting serum levels of IFN-γ, measured 5 hafter injection of different amounts of IL18BP (0; 0.4; 4 mg/kg), whichwas administered 20 min before LPS injection into P. acnes sensitizedmice.

FIG. 9 shows the survival of mice injected either with polyclonal IL-18antiserum or normal rabbit serum (NDS=control) 30 min before injectionwith 40 mg/ml (lethal dosis) of LPS derived from E. coli (FIG. 9 A) orS. thyphimurium (FIG. 9 B). Triangles: mice were injected with IL-18antiserum; circles: mice were injected with NDS. On the x-axis, the daysafter LPS challenge are depicted. * p<0.05.

FIG. 10 shows a histogram, in which the mean±SEM of five mice per grouptreated in the following way are depicted. Mice were injectedintraperitoneally (i.p.) either anti-IL-18 antiserum, soluble TNF-αreceptors (TNFsRp55) or vehicle (saline), immediately followed by theintravenous (i.v.) administration of Concanavalin A (Con A; FIG. 10 A)or PEA (Pseudomonas aeruginosa, FIG. 10 B). **p<0.01; ***p<0.001 vs.ConA or PEA alone; # p<0.01 vs. either TNFsRp55 or anti-IL-18 factorialANOVA.

FIG. 11 shows the effect of IL-18BP on clinical scores in a murine modelof arthritis

-   -   (A) shows a diagram depicting the clinical scores measured after        daily administration of different amounts of IL-18BP or IFN-β or        vehicle (NaCl) i.p. (intraperitoneally) to mice. Symbols: Filled        triangles: 10 000 IU IFN-β; open triangles: 10 mg/kg IL-18BP,        reversed triangles: 3 mg/kg IL-18BP, diamonds: 1 mg/kg IL-18BP;        circles: 0.5 mg/kg IL-18BP; open squares: 0.25 mg/kg IL-18BP,        and filled squares: NaCl. The days of treatment are depicted on        the x-axis, the clinical scores (mean values) are depicted on        the y-axis. Statistics were calculated by the Mann Whitney test.    -   (B) shows a histogram depicting the AUC (area under the curve)        derived from the graph of FIG. 11A. n=number of animals.

FIG. 12 shows the effect of IL-18BP on paw swelling

-   -   (A) shows a diagram depicting the results obtained by measuring        the paw thickness (swelling) of diseased hind paws of individual        animals treated with different amounts of IL-18BP. The y-axis        shows the change of paw thickness in millimeters from the        beginning of treatment. The symbols are as in FIG. 11.    -   (B) shows a histogram depicting the AUC derived from FIG. 11A.        n=number of animals.

FIG. 13 shows the analysis of the number of diseased hind paws at thetime of acute arthritis, i.e. spreading of the disease to additionaljoints. Symbols: Filled squares: NaCl (control), triangles: 10 mg/kgIL-18BP, reversed triangles: 3 mg/kg IL-18BP, diamonds: 1 mg/kg IL-18BP,circles: 0.5 mg/kg IL-18BP and open squares: 0.25 mg/kg IL-18BP.

FIG. 14 shows a histogram depicting the erosion scores of the cartilageof diseased joints.

FIG. 15 shows the histopathology of mouse joints. At the end of theexperiment, the paw that first developed arthritis was dissected away,fixed and processed as described in Example 2 below. A) normal mousejoint; B) joint from an arthritic mouse; C) joint from a mouse treatedwith rhIL-18BP.

FIG. 16 shows a histogram depicting the levels of anti-collagen type IIantibodies of the isotype IgG1 (open columns) or IgG2a (hatched columns)of mice treated with 3 mg/kg of IL-18BP or saline (vehicle),respectively. Measurements were taken on day 4 (D4) or day 8 (D8) of thedisease.

FIG. 17 shows a histogram depicting IL-6 levels in pg/ml of animalstreated with 1, 3 or 10 mg/kg of IL-18BP, 10 000 IU of Interferon β(IFN-b), normal mouse serum (NMS) or saline (NaCl), respectively.

FIG. 18 shows the distribution of serum IL-18BPa in healthy individuals.Sera of male and female healthy individuals ages 20-60, as assayed forthe presence of IL-18BPa by a specific ELISA test (n=107).

FIG. 19A shows the average levels of IL-18 and IL-18BPa in sepsispatients and healthy subjects. Serum IL-18 and IL-18BPa were determinedin the healthy individuals (see FIG. 18) and in 198 samples from 42sepsis patients immediately upon hospital admission and duringhospitalisation.

FIG. 19 B shows the distribution of the individual levels of IL-18 andIL-18BPa of the healthy subjects and patients described in FIG. 19A. Themean serum levels of IL-18 and IL-18BPa in healthy subjects areindicated by a dashed vertical and horizontal line, respectively.

FIG. 20 shows a comparison between the total and free IL-18 inindividual sepsis patients upon hospital admission. The level of freeIL-18 (closed circles) in sera was calculated based on the concentrationof total IL-18 (open circles) and IL-18BPa, taking into account astoichiometry of 1:1 of IL-18 to IL-18BPa in a complex and a calculatedKd of 400 μM. Each vertical line links total and free IL-18 in anindividual serum sample.

FIG. 21 shows the effect of IL-18BP on S. epidermidis-induced IFN-γproduction in whole blood. Whole blood was stimulated with S.epidermidis alone or S. epidermidis and recombinant IL-18BP at indicatedconcentrations. After 48 hours of incubation, the blood culture waslysed and IFN-γ was measured. The results are expressed as percentage ofS. epidermidis IFN-γ induction. P<0.01 vs. S. epidermidis alone. Thedata represents the means±standard error of the mean (SEM) of sixexperiments. SEM represents the spread of the mean of a sample. SEMgives an idea of the accuracy of the mean. SEM=SD/(square root of samplesize). The data was analysed with paired Student's test.

FIG. 22 shows the inhibitory effect exerted by the double activity ofIL-18BPa and anti IL-12 monoclonal antibodies on the production of IFN-γby whole blood treated with S. epidermidis. Whole blood was stimulatedwith S. epidermidis in the presence or in the absence or of IL-18BPa(125 ng/ml), IL-12 Mab (2.5 μg/ml) and both. After 48 hours ofincubation, the blood culture was lysed and IFN-γ was measured. The datais expressed as percent of IFN-γ induction. P<0.01 vs. S. epidermidisalone. The data represents the mean±standard error of the mean (SEM) ofsix experiments. SEM represents the spread of the mean of a sample. SEMgives an idea of the accuracy of the mean. SEM=SD/(square root of samplesize). The data was analysed with paired Student's test.

FIG. 23 shows the quantitation of IL-18BPa as reflected in an ELISAstandard curve. Recombinant human IL-18BPa was serially diluted andsubjected to ELISA as described in Example 30. The data shows the meanabsorbance±SE (standard error) of 10 experiments.

FIG. 24 shows the inhibitory effect of IL-18 on the quantitation ofIL-18BPa by ELISA assay. The percent of inhibition of the signal isshown.

FIG. 25 shows the immunological cross reactivity of human IL-18BPisoforms. The IL-18P ELISA was performed with all isoforms (a-d) ofIL-18BP. Stocks (3.2 μg/ml) of the IL-18BP isoforms were seriallydiluted and tested in IL-18BPa ELISA.

FIG. 26 shows myocardial tissue content of IL-18 following LPSadministration. Mice were injected with E. coli LPS (0.5 mg/Kg,intraperitonially). Hearts were homogenized at the indicated times atthe x axis. ELISA was carried out to determine myocardial IL-18 content.(n=4-5 per group).

FIG. 27 shows the effect of anti IL-18 antibody on LPS-inducedmyocardial dysfunction. Mice were injected with either vehicle(intraperitoneally injected saline n=8) or E. coli LPS (0.5 mg/Kginjected intraperitoneally, n=8) and the left ventricular developedpressure (LVDP) was determined at 6 hours by isolated heart perfusion.In separate experiments, mice were pretreated with either normal rabbitserum (NRS, n=5) or anti-IL-18 antibody (Anti IL-18, n=8) 30 minutesprior to LPS.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to various IL-18BPs and viral IL-18BPswhich bind to IL-18 and thus, are capable of blocking the biologicalactivities of IL-18. The term, “IL-18BPs and viral IL-18BPs,” includesmuteins of IL-18BPs and viral IL-18BPs, derivatives of IL-18BPs andviral IL-18BPs and truncated forms of IL-18BPs and viral IL-18BPs andsalts thereof. The invention further relates to replicable expressionvehicles, suitable for expression of various IL-18BPs or viral IL-18BPsin host cells and host bacteria. The invention further relates toexpression vectors, suitable for expression of various IL-18BPs or viralIL-18BPs in humans and in other mammals.

The replicable expression vehicles containing DNAs suitable forexpression of various IL-18BPs and viral IL-18BPs in host cells,according to the invention, may be PCR products, cDNA, synthetic DNA andcombinations thereof. DNA molecules hybridizing to the above DNAs understringent conditions and encoding proteins or polypeptides having thesame activity as IL-18BP are also included in the present invention.

The expression vectors, suitable for expression of various IL-18BPs orviral IL-18BPs in humans and in other mammals, according to the presentinvention, may be viral vectors or other types of vectors to which anIL-18BP gene or an IL-18BP cDNA or a DNA encoding a viral IL-18BP wasinserted in a way that enables efficient expression of an IL-18BP or aviral IL-18BP in humans and other mammals. DNA molecules hybridizing tothe above DNAs under stringent conditions and encoding proteins orpolypeptides having the same activity as IL-18BP, are also included inthe present invention.

Isolation of IL-18BP may be accomplished in accordance with theinvention, e.g. by passing a human fluid, such as urine or serum,through a chromatographic column to which IL-18 is coupled, andthereafter, eluting the bound IL-18BP.

The various IL-18BPs and viral IL-18BPs can also be prepared byrecombinant means, i.e. by expressing IL-18BP in a suitable host, afteradding promoters, expression enhancers, etc., suitable for theparticular host employed.

The various IL-18BPs and viral IL-18BPs and vectors for expressing IL-18Bp in humans and other mammals may be employed in the treatment andalleviation of conditions in which IL-18 is involved or caused by anexcess of exogenously administered or endogenously produced IL-18. Suchconditions are, e.g., autoimmune diseases, type I diabetes, rheumatoidarthritis, graft rejections, inflammatory bowel disease, sepsis,multiple sclerosis, ischemic heart diseases (including heart attacks),ischemic brain injury, chronic hepatitis, psoriasis, chronicpancreatitis, acute pancreatitis and the like.

According to the present invention, IL-18BP was isolated from normalhuman urine by one chromatographic step. A preparation of crude humanurinary proteins was loaded on a column consisting of human IL-18 boundto agarose. The column was washed and bound proteins were eluted at lowpH. Eluted fractions were neutralized and aliquots were analyzed bySDS-PAGE (10% acrylamide) under non-reducing conditions and silverstaining. A protein band of ˜40 kD was specifically obtained in theeluted fractions (FIG. 1).

The ˜40 kD protein obtained in the first step was identified as an IL-18binding protein by its ability to specifically cross-link with¹²⁵I-IL-18 (FIG. 2). The ˜40 kD protein was further characterized byN-terminal protein sequence analysis. Aliquots from the eluted proteinwere subjected to SDS-PAGE, electroblotted to a PVDF membrane andsubjected to protein microsequence analysis. Similarly, aliquots fromthe eluted protein were subjected to direct protein microsequenceanalysis. In both cases, two polypeptide sequences were obtained. Amajor sequence and a minor sequence, the latter corresponding to afragment of human defensin (accession number p11398), starting at aminoacid 65. Subtraction of the known defensin sequence provided thefollowing new sequence:

T-P-V-S-Q-Q-x-x-x-A-A-A- (SEQ ID NO: 11) 1 . . . 5 . . . . 10. .

wherein x represents a yet undetermined amino acid.

In order to obtain a longer and more accurate sequence and in order toidentify potential cysteine residues, an aliquot of the eluted fractionwas reduced with DTT under denaturing conditions, reacted with 4-vinylpyridine, desalted by a micro-ultrafiltration device (Ultrafree, cutoff10,000 Da, Millipore) and subjected to protein microsequence analysis.After sequencing cycle No. 1 the residual protein was reacted witho-phtalaldehyde to block all N-terminal polypeptides other than Pro andsequencing was then resumed. In this way the following single proteinsequence was obtained:

(SEQ ID NO: 10) TPVSQXXXAA XASVRSTKDP CPSQPPVFPA AKQCPALEVT1       10         20         30         40 (T = Thr; P = Pro; V = Val;S = Ser; Q = Gln; X = Unknown; A = Ala; R = Arg; K = Lys; D = Asp; C =Cys; F = Phe; L = Leu; E = Glu)

The resulting sequence is significantly different from that of any otherknown protein, as determined by searching protein databases. However,searching the database of The Institute of Genomic Research (TIGR) bythe tblastn search program provided a cDNA file, denoted THC123801,whose open reading frame (218 codons) contains a sequence highlyhomologous to that of the N-terminal sequence of IL-18BP. The homologyis hereby shown:

(The upper sequence (1-40) (SEQ ID NO: 12) is that of the isolatedIL-18BP; the lower sequence (51-100) (SEQ ID NO: 13) is derived bytranslation of TIGR file THC123801).

The affinity-purified urinary IL-18BP retained the ability to bind itslabeled ligand (¹²⁵I-IL-18), and following covalent cross-linking, acomplex of molecular weight 58 kD was formed. The molecular weight ofthis complex corresponded to a 1:1 ratio of the ˜40 kD IL-18BP and the19 kD IL-18 (FIG. 2).

The affinity-purified urinary IL-18BP blocked the biological activity ofhuman as well as mouse IL-18. Thus when IL-18BP was added to eitherhuman or mouse IL-18 it blocked the ability of IL-18 to induce theproduction of interferon-γ when added together with lipopolysaccharideto cultures of mouse spleen cells (FIG. 3).

Using the partial sequence of the IL-18BP cDNA as provided by the TIGRdatabase, a DNA probe was prepared by reverse-transcription PCR withspecific sense and antisense primers and RNA from the human Jurkat Tcells. The resulting PCR product was confirmed by DNA sequence analysis.This PCR product was labeled with ³²[P] and used as a probe forscreening of four human cDNA libraries, derived from peripheral bloodmonocytes, from the Jurkat T-cell line, from PBMC and from human spleen.The various independent cDNA clones corresponded to four IL-18BP splicevariants. All splice variants coded for putative soluble secretedproteins. The most abundant one (IL-18BPa) had an open reading frame of192 codons, coding for a signal peptide of 28 amino acid residuesfollowed by a mature putative IL-18BPa, whose first 40 residues matchedperfectly with the N-terminal protein sequence of the urinary IL-18BP(SEQ ID NO:1 and SEQ ID NO:2). The position of the cysteine residuessuggested that this polypeptide belongs to the immunoglobulin (Ig)super-family. Interestingly, each of the four Gln residues within matureIL-18BPa was a potential N-glycosylation site. The three other variantsof IL-18BP were less abundant than IL-18BPa. They included a shorter 1kb IL-18BPb cDNA, coding for a signal peptide of 28 amino acid residuesfollowed by a mature protein of 85 amino acid residues (SEQ ID NO:3 andSEQ ID NO:4). A third variant, IL-18BPc was represented by a 2.3 kbcDNA, coding for a signal peptide of 28 amino acid residues followed bya mature IL-18BP of 169 amino acid residues (SEQ ID NO:5 and SEQ IDNO:6). The fourth variant, IL-18BPd, coded for a signal peptide of 28amino acid residues followed by a mature IL-18BP of 133 amino acidresidues (SEQ ID NO:7 and SEQ ID NO:8).

To further study the possible existence of additional IL-18BP splicevariants, a human genomic library was screened with a probecorresponding to full length IL-18BP cDNA. Five genomic clones,differing in length, were identified in this library. These clones weresubjected to DNA sequence analysis with external and internal primers.Altogether, a 7.1 kb contig was assembled from these clones (SEQ IDNO:9). No exon coding for a trans-membrane (TM) receptor was identifiedwithin the 7.1 kb contig. All variants shared a common translation startsite, coded for the same signal peptide of 28 amino acid residues andsoluble mature proteins of varying sizes and C-terminal sequences. TheIL-18BP locus contains an additional gene, coding for the nuclearmitotic apparatus protein 1 (NUMA1), positioned at the minus strand.This finding localizes the IL-18BP gene to human chromosome 11q13(Broach et al., 1981).

An homology search was done with the complete protein sequence ofIL-18BPa and the GenPept database, using the Smith Watermann algorithm.It was found that homologues of IL-18BP are expressed in severalPoxviruses as secreted proteins of a previously unknown function. It waspreviously reported that viruses code for various cytokine receptors andthat such virally encoded molecules serve as decoy receptors thatinhibit immune responses by neutralizing their corresponding cytokine(reviewed by Spriggs, M K, 1994, Curr. Opin. Immunol., 6, 526-529).Therefore the invention further relates to virally encoded homologues ofIL-18BP that may also serve as blockers of the biological activity ofIL-18. Examples of virus-encoded homologues of IL-18BP are provided inTable 1.

TABLE 1 Virus-encoded proteins, showing high homology to human IL-18BPGenPept sequence Virus type MCU60315_54 U60315 Molluscum contagiosumvirus subtype 1 MCU60315_53 U60315 Molluscum contagiosum virus subtype 1SWPHLSB_12 L22013 Swinepox virus CV41KBPL_14 Cowpox virus VVCGAA_5Variola virus U01161_3 174 Ectromelia virus (mouse Poxvirus) VVU18340_6Variola virus VVU18338_7 Variola virus VVU18337_7 Variola virus VARCG_7173 Variola major virus MCU60315_51 Molluscum contagiosum virus HNABV_1New Hepatitis non-A, non-B associated virus

IL-18BPa was expressed in monkey COS7 cells. For this purpose, the cDNAof IL-18BPa was inserted into the mammalian expression vector pEF-BOS. Acassette coding for an (His)₆ sequence was added to the 3′-end of theIL-18BP ORFs in frame, in order to facilitate purification of therecombinant protein. COS7 cells were transiently transfected with theexpression vector and serum-free medium of these cells (150 ml) wasconcentrated and purified by metal chelate chromatography. IL-18BPa ranas a single band upon SDS-PAGE with silver staining under reducing andnon-reducing conditions and had the same apparent molecular mass as thatof the urinary IL-18BP. Protein sequence analysis of this preparationrevealed the same N-terminal sequence as that of the urinary IL-18BP.Immunoblot analysis of IL-18BPa with antibodies raised against theurinary IL-18BP revealed the same molecular mass band as that of theurinary protein. Furthermore, using immunoprecipitation followed bySDS-PAGE and autoradiography, IL-18BPa was able to displace urinary¹²⁵I-IL-18BP from binding to the antibody. Therefore, IL-18BPacorresponds structurally to the IL-18BP isolated from urine.

Crude and purified IL-18BPa were tested for their ability to inhibit thebiological activity of IL-18. IL-18BPa inhibited the activity of humanand mouse IL-18 in murine splenocytes, PBMC and the human KG-1 cell line(FIG. 4). In contrast, IL-18BPb did not significantly inhibit theactivity of IL-18. These results confirm the identity of IL-18BPa cDNAas the one coding for a biologically active IL-18BP.

The invention further relates to active muteins and fragments ofIL-18BPs and viral IL-18BPs and to fused proteins consisting of wildtype IL-18BPs and viral IL-18BPs or their active muteins or their activefragments, fused to another polypeptide or protein and exhibiting asimilar ability to block the biological activities of IL-18 or itshomologues.

As used herein the term “muteins” refers to analogs of an IL-18BP, oranalogs of a viral IL-18BP, in which one or more of the amino acidresidues of a natural IL-18BP or viral IL-18BP are replaced by differentamino acid residues, or are deleted, or one or more amino acid residuesare added to the natural sequence of an IL-18BP, or a viral IL-18BP,without changing considerably the activity of the resulting products ascompared with the wild type IL-18BP or viral IL-18BP. These muteins areprepared by known synthesis and/or by site-directed mutagenesistechniques, or any other known technique suitable therefor.

Any such mutein preferably has a sequence of amino acids sufficientlyduplicative of that of an IL-18BP, or sufficiently duplicative of aviral IL-18BP, such as to have substantially similar activity toIL-18BP. One activity of IL-18BP is its capability of binding IL-18. Aslong as the mutein has substantial binding activity to IL-18, it can beused in the purification of IL-18, such as by means of affinitychromatography, and thus can be considered to have substantially similaractivity to IL-18BP. Thus, it can be determined whether any given muteinhas substantially the same activity as IL-18BP by means of routineexperimentation comprising subjecting such a mutein, e.g., to a simplesandwich competition assay to determine whether or not it binds to anappropriately labeled IL-18, such as radioimmunoassay or ELISA assay.

In a preferred embodiment, any such mutein has at least 40% identity orhomology with the sequence of either an IL-18BP or a virally-encodedIL-18BP homologue. More preferably, it has at least 50%, at least 60%,at least 70%, at least 80% or, most preferably, at least 90% identity orhomology thereto.

Muteins of IL-18BP polypeptides or muteins of viral IL-18BPs, which canbe used in accordance with the present invention, or nucleic acid codingtherefor, include a finite set of substantially corresponding sequencesas substitution peptides or polynucleotides which can be routinelyobtained by one of ordinary skill in the art, without undueexperimentation, based on the teachings and guidance presented herein.For a detailed description of protein chemistry and structure, seeSchulz, G. E. et al., Principles of Protein Structure, Springer-Verlag,New York, 1978; and Creighton, T. E., Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, 1983, which are herebyincorporated by reference. For a presentation of nucleotide sequencesubstitutions, such as codon preferences, see Ausubel et al, supra, at§§ A.1.1-A.1.24, and Sambrook et al, supra, at Appendices C and D.

Preferred changes for muteins in accordance with the present inventionare what are known as “conservative” substitutions. Conservative aminoacid substitutions of IL-18BP polypeptides or proteins or viralIL-18BPs, may include synonymous amino acids within a group which havesufficiently similar physicochemical properties that substitutionbetween members of the group will preserve the biological function ofthe molecule, Grantham, Science, Vol. 185, pp. 862-864 (1974). It isclear that insertions and deletions of amino acids may also be made inthe above-defined sequences without altering their function,particularly if the insertions or deletions only involve a few aminoacids, e.g., under thirty, and preferably under ten, and do not removeor displace amino acids which are critical to a functional conformation,e.g., cysteine residues, Anfinsen, “Principles That Govern The Foldingof Protein Chains”, Science, Vol. 181, pp. 223-230 (1973). Proteins andmuteins produced by such deletions and/or insertions come within thepurview of the present invention.

Preferably, the synonymous amino acid groups are those defined in TableI. More preferably, the synonymous amino acid groups are those definedin Table 2; and most preferably the synonymous amino acid groups arethose defined in Table 31.

TABLE 1 Preferred Groups of Synonymous Amino Acids Amino Acid SynonymousGroup Ser Ser, Thr, Gly, Asn Arg Arg, Gln, Lys, Glu, His Leu Ile, Phe,Tyr, Met, Val, Leu Pro Gly, Ala, Thr, Pro Thr Pro, Ser, Ala, Gly, His,Gln, Thr Ala Gly, Thr, Pro, Ala Val Met, Tyr, Phe, Ile, Leu, Val GlyAla, Thr, Pro, Ser, Gly Ile Met, Tyr, Phe, Val, Leu, Ile Phe Trp, Met,Tyr, Ile, Val, Leu, Phe Tyr Trp, Met, Phe, Ile, Val, Leu, Tyr Cys Ser,Thr, Cys His Glu, Lys, Gln, Thr, Arg, His Gln Glu, Lys, Asn, His, Thr,Arg, Gln Asn Gln, Asp, Ser, Asn Lys Glu, Gln, His, Arg, Lys Asp Glu,Asn, Asp Glu Asp, Lys, Asn, Gln, His, Arg, Glu Met Phe, Ile, Val, Leu,Met Trp Trp

TABLE 2 More Preferred Groups of Synonymous Amino Acids Amino AcidSynonymous Group Ser Ser Arg His, Lys, Arg Leu Leu, Ile, Phe, Met ProAla, Pro Thr Thr Ala Pro, Ala Val Val, Met, Ile Gly Gly Ile Ile, Met,Phe, Val, Leu Phe Met, Tyr, Ile, Leu, Phe Tyr Phe, Tyr Cys Cys, Ser HisHis, Gln, Arg Gln Glu, Gln, His Asn Asp, Asn Lys Lys, Arg Asp Asp, AsnGlu Glu, Gln Met Met, Phe, Ile, Val, Leu Trp Trp

TABLE 3 Most Preferred Groups of Synonymous Amino Acids Amino AcidSynonymous Group Ser Ser Arg Arg Leu Leu, Ile, Met Pro Pro Thr Thr AlaAla Val Val Gly Gly Ile Ile, Met, Leu Phe Phe Tyr Tyr Cys Cys, Ser HisHis Gln Gln Asn Asn Lys Lys Asp Asp Glu Glu Met Met, Ile, Leu Trp Met

Examples of production of amino acid substitutions in proteins which canbe used for obtaining muteins of IL-18BP polypeptides or proteins, ormuteins of viral IL-18BPs, for use in the present invention include anyknown method steps, such as presented in U.S. Pat. Nos. RE 33,653,4,959,314, 4,588,585 and 4,737,462, to Mark et al; 5,116,943 to Koths etal., 4,965,195 to Namen et al; 4,879,111 to Chong et al; and 5,017,691to Lee et al; and lysine substituted proteins presented in U.S. Pat. No.4,904,584 (Shaw et al).

In another preferred embodiment of the present invention, any mutein ofan IL-18BP or a viral IL-18BP, has an amino acid sequence essentiallycorresponding to that of an IL-18BP, or to a viral IL-18BP. The term“essentially corresponding to” is intended to comprehend proteins withminor changes to the sequence of the natural protein which do not affectthe basic characteristics of the natural proteins, particularly insofaras their ability to bind IL-18 and to thereby inhibit its biologicalactivity. The type of changes which are generally considered to fallwithin the “essentially corresponding to” language are those which wouldresult from conventional mutagenesis techniques of the DNA encodingthese proteins, resulting in a few minor modifications, and screeningfor the desired activity in the manner discussed above. Muteins inaccordance with the present invention include proteins encoded by anucleic acid, such as DNA or RNA, which hybridizes to DNA or RNA, whichencodes an IL-18BP or encodes a viral IL-18BP, in accordance with thepresent invention, under stringent conditions. The invention alsoincludes such nucleic acid, which is also useful as a probe inidentification and purification of the desired nucleic acid.Furthermore, such nucleic acid would be a prime candidate to determinewhether it encodes a polypeptide, which retains the functional activityof an IL-18BP of the present invention. The term “stringent conditions”refers to hybridization and subsequent washing conditions, which thoseof ordinary skill in the art conventionally refer to as “stringent”. SeeAusubel et al., Current Protocols in Molecular Biology, supra,Interscience, N.Y., §§6.3 and 6.4 (1987, 1992), and Sambrook et al.,supra. For example high stringent conditions include washing conditions12-20° C. below the calculated Tm of the hybrid under study in, e.g.,2×SSC and 0.5% SDS for 5 minutes, 2×SSC and 0.1% SDS for 15 minutes;0.1×SSC and 0.5% SDS at 37° C. for 30-60 minutes and then, a 0.1×SSC and0.5% SDS at 68° C. for 30-60 minutes. Those of ordinary skill in thisart understand that stringency conditions also depend on the length ofthe DNA sequences, oligonucleotide probes (such as 10-40 bases) or mixedoligonucleotide probes. If mixed probes are used, it is preferable touse tetramethyl ammonium chloride (TMAC) instead of SSC. See Ausubel,supra.

The term “fused protein” refers to a polypeptide comprising an IL-18BP,or a viral IL-18BP, or a mutein thereof, fused with another protein,which, e.g., has an extended residence time in body fluids. An IL-18BPor a viral IL-18BP, may thus be fused to another protein, polypeptide orthe like, e.g., an immunoglobulin or a fragment thereof.

The term “salts” herein refers to both salts of carboxyl groups and toacid addition salts of amino groups of an IL-18BP, a viral IL-18BP,muteins, or fused proteins thereof. Salts of a carboxyl group may beformed by means known in the art and include inorganic salts, forexample, sodium, calcium, ammonium, ferric or zinc salts, and the like,and salts with organic bases as those formed, for example, with amines,such as triethanolamine, arginine or lysine, piperidine, procaine andthe like. Acid addition salts include, for example, salts with mineralacids such as, for example, hydrochloric acid or sulfuric acid, andsalts with organic acids such as, for example, acetic acid or oxalicacid. Of course, any such salts must have substantially similar activityto IL-18BP.

“Functional derivatives” as used herein cover derivatives of IL-18BPs ora viral IL-18BP, and their muteins and fused proteins, which may beprepared from the functional groups which occur as side chains on theresidues or the N- or C-terminal groups, by means known in the art, andare included in the invention as long as they remain pharmaceuticallyacceptable, i.e. they do not destroy the activity of the protein whichis substantially similar to the activity of IL-18BP, or viral IL-18BPs,and do not confer toxic properties on compositions containing it. Thesederivatives may, for example, include polyethylene glycol side-chains,which may mask antigenic sites and extend the residence of an IL-18BP ora viral IL-18BP in body fluids. Other derivatives include aliphaticesters of the carboxyl groups, amides of the carboxyl groups by reactionwith ammonia or with primary or secondary amines, N-acyl derivatives offree amino groups of the amino acid residues formed with acyl moieties(e.g. alkanoyl or carbocyclic aroyl groups) or O-acyl derivatives offree hydroxyl groups (for example that of seryl or threonyl residues)formed with acyl moieties.

As “active fractions” of an IL-18BP, or a viral IL-18BP, muteins andfused proteins, the present invention covers any fragment or precursorsof the polypeptide chain of the protein molecule alone or together withassociated molecules or residues linked thereto, e.g., sugar orphosphate residues, or aggregates of the protein molecule or the sugarresidues by themselves, provided said fraction has substantially similaractivity to IL-18BP.

Various recombinant cells such as prokaryotic cells, e.g., E. coli, orother eukaryotic cells, such as yeast or insect cells can produceIL-18BPs or viral IL-18BPs. Methods for constructing appropriatevectors, carrying DNA that codes for an IL-18BP and suitable fortransforming (e.g., E. coli, mammalian cells and yeast cells), orinfecting insect cells in order to produce a recombinant IL-18BP or aviral IL-18BP are well known in the art. See, for example, Ausubel etal., eds. “Current Protocols in Molecular Biology” Current Protocols,1993; and Sambrook et al., eds. “Molecular Cloning: A LaboratoryManual”, 2nd ed., Cold Spring Harbor Press, 1989.

Cells that produce IL-18 BPs or viral IL-18BPs are made according toknown procedures in the art, including for example, by gene activation(See, PCT publication WO90/11354, U.S. Pat. Nos. 5,272,071, and5,641,670, which are incorporated herein by reference in theirentirety), and transduction.

For the purposes of expression of IL-18BP proteins, or viral IL-18BPs,DNA encoding an IL-18BP or a viral IL-18BP, their fragments, muteins orfused proteins, and the operably linked transcriptional andtranslational regulatory signals, are inserted into eukaryotic vectorswhich are capable of integrating the desired gene sequences into thehost cell chromosome. In order to be able to select the cells which havestably integrated the introduced DNA into their chromosomes, one or moremarkers which allow for selection of host cells which contain theexpression vector is used. The marker may provide for prototrophy to anauxotropic host, biocide resistance, e.g., antibiotics, or resistance toheavy metals, such as copper, or the like. The selectable marker genecan either be directly linked to the DNA gene sequences to be expressed,or introduced into the same cell by cotransfection. Additional elementsmay also be needed for optimal synthesis of single chain binding proteinmRNA. These elements may include splice signals, as well astranscription promoters, enhancers, and termination signals.

Said DNA molecule to be introduced into the cells of choice willpreferably be incorporated into a plasmid or viral vector capable ofautonomous replication in the recipient host. Preferred prokaryoticplasmids are derivatives of pBr322. Preferred eukaryotic vectors includeBPV, vaccinia, SV40, 2-micron circle, etc., or their derivatives. Suchplasmids and vectors are well known in the art (Anderson, 1997; Bolton,1980; Botstein, 1982 Broach, 1981; Kendall et al., 1987). Once thevector or DNA sequence containing the construct(s) has been prepared forexpression, the expression vector may be introduced into an appropriatehost cell by any of a variety of suitable means, such as transformation,transfection, lipofection, conjugation, protoplast fusion,electroporation, calcium phosphate precipitation, direct microinjection,etc.

Host cells to be used in this invention may be either prokaryotic oreukaryotic. Preferred prokaryotic hosts include bacteria such as E.coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, etc.The most preferred prokaryotic host is E. coli. Bacterial hosts ofparticular interest include E. coli K12 strain 294 (ATCC 31446), E. coliX1776 (ATCC 31537), E. coli W3110 (F⁻, lambda⁻, phototropic (ATCC27325). Under such conditions, the protein will not be glycosylated. Theprokaryotic host must be compatible with the replicon and controlsequences in the expression plasmid.

However, since IL-18BPs are glycosylated proteins, eukaryotic hosts arepreferred over prokaryotic hosts. Preferred eukaryotic hosts aremammalian cells, e.g., human, monkey, mouse and Chinese hamster ovary(CHO) cells, because they provide post-translational modifications toprotein molecules including correct folding, correct disulfide bondformation, as well as glycosylation at correct sites. Also yeast cellsand insect cells can carry out post-translational peptide modificationsincluding high mannose glycosylation.

A number of recombinant DNA strategies exist which utilize strongpromoter sequences and high copy number of plasmids, which can beutilized for production of the desired proteins in yeast and in insectcells. Yeast and insect cells recognize leader sequences on clonedmammalian gene products and secrete mature IL-18BP. After theintroduction of the vector, the host cells are grown in a selectivemedium, which selects for the growth of vector-containing cells.Expression of the cloned gene sequence(s) results in the production ofan IL-18BP, a viral IL-18BP, fusion proteins, or muteins or fragmentsthereof. The above-mentioned cloning, clone isolation, identification,characterization and sequencing procedures are described in more detailhereinafter in the Examples.

The expressed proteins are then isolated and purified by anyconventional procedure involving extraction, precipitation,chromatography, electrophoresis, or the like, or by affinitychromatography, using, e.g., an anti-IL-18BP monoclonal antibodiesimmobilized on a gel matrix contained within a column. Crudepreparations containing said recombinant IL-18BP are passed through thecolumn whereby IL-18BP will be bound to the column by the specificantibody, while the impurities will pass through. After washing, theprotein is eluted from the gel under conditions usually employed forthis purpose, i.e. at a high or a low pH, e.g. pH 11 or pH 2.

The invention further relates to vectors useful for expression of anIL-18BP or a viral IL-18BP or their derivatives in mammals and morespecifically in humans. Vectors for short and long-term expression ofgenes in mammals are well known in the literature. Studies have shownthat gene delivery to e.g., skeletal muscle, vascular smooth muscle andliver result in systemic levels of therapeutic proteins. Skeletal muscleis a useful target because of its large mass, vascularity andaccessibility. However, other targets and particularly bone marrowprecursors of immune cells have been used successfully. Currentlyavailable vectors for expression of proteins in e.g., muscle includeplasmid DNA, liposomes, protein-DNA conjugates and vectors based onadenovirus, adeno-associated virus and herpes virus. Of these, vectorsbased on adeno-associated virus (AAV) have been most successful withrespect to duration and levels of gene expression and with respect tosafety considerations (Kessler, P. D. 1996, Proc. Natl. Acad. Sci. USA93, 14082-14087).

Procedures for construction of an AAV-based vector have been describedin detail (Snyder et al, 1996, Current Protocols in Human Genetics,Chapters 12.1.1-12.1.17, John Wiley & Sons) and are incorporated intothis patent. Briefly plasmid psub201, containing the wild-type AAVgenome is cut with the restriction enzyme Xba I and ligated with aconstruct consisting of an efficient eukaryotic promoter, e.g., thecytomegalovirus promoter, a Kozak consensus sequence, a DNA sequencecoding for an IL-18BP or a viral IL-18BP, or their muteins or fusionproteins or fragments thereof, a suitable 3′ untranslated region and apolyadenylation signal, e.g., the polyadenylation signal of simian virus40. The resulting recombinant plasmid is cotransfected with an helperAAV plasmid e.g., pAAV/Ad into mammalian cells e.g., human T293 cells.The cultures are then infected with adenovirus as a helper virus andculture supernatants are collected after 48-60 hours. The supernatantsare fractionated by ammonium sulfate precipitation, purified on a CsCldensity gradient, dialyzed and then heated at 56° C. to destroy anyadenovirus, whereas the resulting recombinant AAV, capable of expressingIL-18BP or a viral IL-18BP, or their muteins or fusion proteins remainsstable at this step.

So far, the physiological role of the soluble cytokine receptors has notbeen established. The soluble receptors bind their specific ligands andin most cases inhibit their biological activity, as was shown, e.g., inthe TNF system (Dao et al., 1996; Engelmann et al., 1989). In very fewcases, e.g., IL-6, the soluble receptor enhances the biologicalactivity. The recombinant soluble TNF receptor, also known as TBP (TNFbinding protein) was found to prevent septic shock in animal models,while soluble forms of IL-1 receptor were found to have profoundinhibitory effects on the development of in vivo alloreactivity in mouseallograft recipients.

Similarly, the IL-18BPs and viral IL-18BPs of the present invention mayfind use as modulators of IL-18 activity, e.g. in type I diabetes, insepsis, in autoimmune diseases, in graft rejections, rheumatoidarthritis, inflammatory bowel disease, sepsis, multiple sclerosis,ischemic heart disease including acute heart attacks, ischemic braininjury, chronic hepatitis, psoriasis, chronic hepatitis and acutehepatitis. It may thus be used, e.g. in any disease in which endogenousproduction or exogenous administration of IL-18 induces the disease oraggravates the situation of the patient.

The present invention further relates to pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and an IL-18BP or aviral IL-18BP of the invention or their active muteins, fused proteinsand their salts, functional derivatives or active fractions thereof.

The present invention further relates to pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and e.g., a viralvector such as any one of said AAV-based viral vectors or another vectorexpressing an IL-18BP or viral IL-18BP or their muteins, fragments orfusion proteins thereof and suitable for administration to humans andother mammals for the purpose of attaining expression in vivo of IL-18BPor a viral IL-18BP or their muteins or fragments or fusion protein ofthe invention.

The pharmaceutical compositions of the invention are prepared foradministration by mixing an IL-18BP or a viral IL-18BP, or theirderivatives, or vectors for expressing same with physiologicallyacceptable carriers, and/or stabilizers and/or excipients, and preparedin dosage form, e.g., by lyophilization in dosage vials. The method ofadministration can be via any of the accepted modes of administrationfor similar agents and will depend on the condition to be treated, e.g.,intravenously, intramuscularly, subcutaneously, by local injection ortopical application, or continuously by infusion, etc. The amount ofactive compound to be administered will depend on the route ofadministration, the disease to be treated and the condition of thepatient. Local injection, for instance, will require a lower amount ofthe protein on a body weight basis than will intravenous infusion.

Accordingly, IL-18BPs, or viral IL-18BPs, or vectors expressing same invivo are indicated for the treatment of autoimmune diseases, Type Idiabetes, rheumatoid arthritis, graft rejections, inflammatory boweldisease, sepsis, multiple sclerosis, ischemic heart disease includingacute heart attacks, ischemic brain injury, chronic hepatitis,psoriasis, chronic pancreatitis and acute pancreatitis and similardiseases, in which there is an aberrant expression of IL-18, leading toan excess of IL-18 or in cases of complications due to exogenouslyadministered IL-18.

The invention also includes antibodies against an IL-18BP or a viralIL-18BP, as well as against their muteins, fused proteins, salts,functional derivatives and active fractions. The term “antibody” ismeant to include polyclonal antibodies, monoclonal antibodies (MAbs),chimeric antibodies, anti-idiotypic (anti-Id) antibodies to antibodiesthat can be labeled in soluble or bound form, as well as fragmentsthereof provided by any known technique, such as, but not limited toenzymatic cleavage, peptide synthesis or recombinant techniques.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen. Amonoclonal antibody contains a substantially homogeneous population ofantibodies specific to antigens, which population contains substantiallysimilar epitope binding sites. MAbs may be obtained by methods known tothose skilled in the art. See, for example Kohler and Milstein, Nature256:495-497 (1975); U.S. Pat. No. 4,376,110; Ausubel et al, eds., supra,Harlow and Lane, ANTIBODIES: A LABORATORY MANUAL, Cold Spring HarborLaboratory (1988); and Colligan et al., eds., Current Protocols inImmunology, Greene Publishing Assoc. and Wiley Interscience, N.Y.,(1992, 1993), the contents of which references are incorporated entirelyherein by reference. Such antibodies may be of any immunoglobulin classincluding IgG, IgM, IgE, IgA, GILD and any subclass thereof. A hybridomaproducing a MAb of the present invention may be cultivated in vitro, insitu or in vivo. Production of high titers of MAbs in vivo or in situmakes this the presently preferred method of production.

Chimeric antibodies are molecules, different portions of which arederived from different animal species, such as those having the variableregion derived from a murine MAb and a human immunoglobulin constantregion. Chimeric antibodies are primarily used to reduce immunogenicityin application and to increase yields in production, for example, wheremurine MAbs have higher yields from hybridomas but higher immunogenicityin humans, such that human/murine chimeric MAbs are used. Chimericantibodies and methods for their production are known in the art(Cabilly et al, Proc. Natl. Acad. Sci. USA 81:3273-3277 (1984); Morrisonet al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984); Boulianne etal., Nature 312:643-646 (1984); Cabilly et al., European PatentApplication 125023 (published Nov. 14, 1984); Neuberger et al., Nature314:268-270 (1985); Taniguchi et al., European Patent Application 171496(published Feb. 19, 1985); Morrison et al., European Patent Application173494 (published Mar. 5, 1986); Neuberger et al., PCT Application WO8601533, (published Mar. 13, 1986); Kudo et al., European PatentApplication 184187 (published Jun. 11, 1986); Morrison et al., EuropeanPatent Application 173494 (published Mar. 5, 1986); Sahagan et al., J.Immunol. 137:1066-1074 (1986); Robinson et al., International PatentPublication, WO 9702671 (published 7 May 1987); Liu et al., Proc. Natl.Acad. Sci. USA 84:3439-3443 (1987); Sun et al., Proc. Natl. Acad. Sci.USA 84:214-218 (1987); Better et al., Science 240:1041-1043 (1988); andHarlow and Lane, ANTIBODIES: A LABORATORY MANUAL, supra. Thesereferences are entirely incorporated herein by reference.

An anti-idiotypic (anti-Id) antibody is an antibody, which recognizesunique determinants generally, associated with the antigen-binding siteof an antibody. An Id antibody can be prepared by immunizing an animalof the same species and genetic type (e.g., mouse strain) as the sourceof the MAb with the MAb to which an anti-Id is being prepared. Theimmunized animal will recognize and respond to the idiotypicdeterminants of the immunizing antibody by producing an antibody tothese idiotypic determinants (the anti-Id antibody). See, for example,U.S. Pat. No. 4,699,880, which is herein entirely incorporated byreference.

The anti-Id antibody may also be used as an “immunogen” to induce animmune response in yet another animal, producing a so-calledanti-anti-Id antibody. The anti-anti-Id may be epitopically identical tothe original MAb, which induced the anti-Id. Thus, by using antibodiesto the idiotypic determinants of a MAb, it is possible to identify otherclones expressing antibodies of identical specificity.

Accordingly, MAbs generated against IL-18BP and related proteins of thepresent invention may be used to induce anti-Id antibodies in suitableanimals, such as BALB/c mice. Spleen cells from such immunized mice areused to produce anti-Id hybridomas secreting anti-Id Mabs. Further, theanti-Id Mabs can be coupled to a carrier such as keyhole limpethemocyanin (KLH) and used to immunize additional BALB/c mice. Sera fromthese mice will contain anti-anti-Id antibodies that have the bindingproperties of the original MAb specific for an IL-18BP epitope orepitopes of a viral IL-18BP.

The anti-Id MAbs thus have their own idiotypic epitopes, or “idiotopes”structurally similar to the epitope being evaluated, such as an IL-18BPor a viral IL-18BP.

The term “antibody” is also meant to include both intact molecules aswell as fragments thereof, such as, for example, Fab and F(ab′)₂, whichare capable of binding antigen. Fab and F(ab′)₂ fragments lack the Fcfragment of intact antibody, clear more rapidly from the circulation,and may have less non-specific tissue binding than an intact antibody(Wahl et al., J. Nucl. Med. 24:316-325 (1983)). It will be appreciatedthat Fab and F(ab′)₂ and other fragments of the antibodies useful in thepresent invention may be used for the detection and quantitation of anIL-18BP or a viral IL-18BP, according to the methods disclosed hereinfor intact antibody molecules. Such fragments are typically produced byproteolytic cleavage, using enzymes such as papain (to produce Fabfragments) or pepsin (to produce F(ab′)₂ fragments).

An antibody is said to be “capable of binding” a molecule if it iscapable of specifically reacting with the molecule to thereby bind themolecule to the antibody. The term “epitope” is meant to refer to thatportion of any molecule capable of being bound by an antibody which canalso be recognized by that antibody. Epitopes or “antigenicdeterminants” usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and have specificthree dimensional structural characteristics as well as specific chargecharacteristics.

An “antigen” is a molecule or a portion of a molecule capable of beingbound by an antibody which is additionally capable of inducing an animalto produce antibody capable of binding to an epitope of that antigen. Anantigen may have one or more than one epitope. The specific reactionreferred to above is meant to indicate that the antigen will react, in ahighly selective manner, with its corresponding antibody and not withthe multitude of other antibodies which may be evoked by other antigens.

The antibodies, including fragments of antibodies, useful in the presentinvention may be used to detect quantitatively or qualitatively anIL-18BP or a viral IL-18BP, or related proteins in a sample or to detectpresence of cells, which express such proteins of the present invention.This can be accomplished by immunofluorescence techniques employing afluorescently labeled antibody (see below) coupled with lightmicroscopic, flow cytometric, or fluorometric detection.

The antibodies (or fragments thereof) useful in the present inventionmay be employed histologically, as in immunofluorescence orimmunoelectron microscopy, for in situ detection of an IL-18BP or aviral IL-18BP, and related proteins of the present invention. In situdetection may be accomplished by removing a histological specimen from apatient, and providing the a labeled antibody of the present inventionto such a specimen. The antibody (or fragment) is preferably provided byapplying or by overlaying the labeled antibody (or fragment) to abiological sample. Through the use of such a procedure, it is possibleto determine not only the presence of an IL-18BP or a viral IL-18BP, orrelated proteins but also its distribution on the examined tissue. Usingthe present invention, those of ordinary skill will readily perceivethat any of wide variety of histological methods (such as stainingprocedures) can be modified in order to achieve such in situ detection.

Such assays for an IL-18BP or a viral IL-18BP, or related proteins ofthe present invention typically comprises incubating a biologicalsample, such as a biological fluid, a tissue extract, freshly harvestedcells such as lymphocytes or leukocytes, or cells which have beenincubated in tissue culture, in the presence of a detectably labeledantibody capable of identifying IL-18BP or related proteins, anddetecting the antibody by any of a number of techniques well-known inthe art.

The biological sample may be treated with a solid phase support orcarrier such as nitrocellulose, or other solid support or carrier whichis capable of immobilizing cells, cell particles or soluble proteins.The support or carrier may then be washed with suitable buffers followedby treatment with a detectably labeled antibody in accordance with thepresent invention. The solid phase support or carrier may then be washedwith the buffer a second time to remove unbound antibody. The amount ofbound label on said solid support or carrier may then be detected byconventional means.

By “solid phase support”, “solid phase carrier”, “solid support”, “solidcarrier”, “support” or “carrier” is intended any support or carriercapable of binding antigen or antibodies. Well-known supports orcarriers, include glass, polystyrene, polypropylene, polyethylene,dextran, nylon amylases, natural and modified celluloses,polyacrylamides, gabbros, and magnetite. The nature of the carrier canbe either soluble to some extent or insoluble for the purposes of thepresent invention. The support material may have virtually any possiblestructural configuration so long as the coupled molecule is capable ofbinding to an antigen or antibody. Thus, the support or carrierconfiguration may be spherical, as in a bead, or cylindrical, as in theinside surface of a test tube, or the external surface of a rod.Alternatively, the surface may be flat such as a sheet, test strip, etc.Preferred supports or carriers include polystyrene beads. Those skilledin the art will know many other suitable carriers for binding antibodyor antigen, or will be able to ascertain the same by use of routineexperimentation.

The binding activity of a given lot of antibody in accordance with thepresent invention may be determined according to well known methods.Those skilled in the art will be able to determine operative and optimalassay conditions for each determination by employing routineexperimentation.

Other such steps as washing, stirring, shaking, filtering and the likemay be added to the assays as is customary or necessary for theparticular situation.

One of the ways in which an antibody in accordance with the presentinvention can be detectably labeled is by linking the same to an enzymeand use in an enzyme immunoassay (EIA). This enzyme, in turn, when laterexposed to an appropriate substrate, will react with the substrate insuch a manner as to produce a chemical moiety which can be detected, forexample, by spectrophotometric, fluorometric or by visual means. Enzymeswhich can be used detectably label the antibody include, but are notlimited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by colorimetricmethods, which employ a chromogenic substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

Detection may be accomplished using any of a variety of otherimmunoassays. For example, by radioactivity labeling the antibodies orantibody fragments, it is possible to detect an IL-18BP or a viralIL-18BP, through the use of a radioimmunoassay (RIA). A good descriptionof RIA maybe found in Laboratory Techniques and Biochemistry inMolecular Biology, by Work, T. S. et al., North Holland PublishingCompany, NY (1978) with particular reference to the chapter entitled “AnIntroduction to Radioimmuno Assay and Related Techniques” by Chard, T.,incorporated by reference herein. The radioactive isotope can bedetected by such means as the use of a gamma counter or a scintillationcounter or by autoradiography.

It is also possible to label an antibody in accordance with the presentinvention with a fluorescent compound. When the fluorescently labeledantibody is exposed to light of the proper wavelength, its presence canbe then be detected due to fluorescence. Among the most commonly usedfluorescent labeling compounds are fluorescein isothiocyanate,rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehydeand fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriamine pentaacetic acid (ETPA). The antibody can also bedetectably labeled by coupling it to biotin. Biotinylated antibody canthen be detected by avidin or streptavidin coupled to a fluorescentcompound or to an enzyme such as peroxidase or to a radioactive isotopeand the like.

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

An antibody molecule of the present invention may be adapted forutilization in a immunometric assay, also known as a “two-site” or“sandwich” assay. In a typical immunometric assay, a quantity ofunlabeled antibody (or fragment of antibody) is bound to a solid supportor carrier and a quantity of detectably labeled soluble antibody isadded to permit detection and/or quantitation of the ternary complexformed between solid-phase antibody, antigen, and labeled antibody.

Typical, and preferred, immunometric assays include “forward” assays inwhich the antibody bound to the solid phase is first contacted with thesample being tested to extract the antigen form the sample by formationof a binary solid phase antibody-antigen complex. After a suitableincubation period, the solid support or carrier is washed to remove theresidue of the fluid sample, including unreacted antigen, if any, andthen contacted with the solution containing an unknown quantity oflabeled antibody (which functions as a “reporter molecule”). After asecond incubation period to permit the labeled antibody to complex withthe antigen bound to the solid support or carrier through the unlabeledantibody, the solid support or carrier is washed a second time to removethe unreacted labeled antibody.

In another type of “sandwich” assay, which may also be useful with theantigens of the present invention, the so-called “simultaneous” and“reverse” assays are used. A “simultaneous” assay involves a singleincubation step as the antibody bound to the solid support or carrierand labeled antibody are both added to the sample being tested at thesame time. After the incubation is completed, the solid support orcarrier is washed to remove the residue of fluid sample and uncomplexedlabeled antibody. The presence of labeled antibody associated with thesolid support or carrier is then determined as it would be in aconventional “forward” sandwich assay.

In the “reverse” assay, stepwise addition first of a solution of labeledantibody to the fluid sample followed by the addition of unlabeledantibody bound to a solid support or carrier after a suitable incubationperiod is utilized. After a second incubation, the solid phase is washedin conventional fashion to free it of the residue of the sample beingtested and the solution of unreacted labeled antibody. The determinationof labeled antibody associated with a solid support or carrier is thendetermined as in the “simultaneous” and “forward” assays.

The present invention also provides DNA molecules encoding any of theproteins of the present invention as defined above, replicableexpression vehicles comprising any such DNA molecules, host cellstransformed with any such expression vehicles including prokaryotic andeukaryotic and host cells, preferably CHO cells. The invention alsoincludes a process for the production of expression vectors coding forany of the proteins of the present invention for the purpose of theirexpression in humans and other mammals.

The invention also includes a process for the production of any of theproteins of the present invention by culturing a transformed cell inaccordance with the present invention and recovering the protein encodedby the DNA molecule and the expression vehicle within such transformedhost cell.

In addition to the use of an IL-18BP or a viral IL-18BP, in modulatingthe activity of IL-18, they can, of course, also be employed for thepurification of IL-18 itself. For this purpose, IL-18BP or a viralIL-18BP is coupled to an affinity column and crude IL-18 is passedthrough. The IL-18 can then be recovered from the column by, e.g.,elution at low pH.

The present invention further relates to the treatment and/or preventionof liver injury using inhibitors of IL-18. An inhibitor according to theinvention can be an inhibitor of IL-18 production and/or action.

The term “inhibitor of IL-18” within the context of this inventionrefers to any molecule modulating IL-18 production and/or action in sucha way that IL-18 production and/or action is attenuated, reduced, orpartially, substantially or completely prevented or blocked.

An inhibitor of production can be any molecule negatively affecting thesynthesis, processing or maturation of IL-18 on the level of the gene,the mRNA or the protein. The inhibitors considered according to theinvention can be, for example, suppressors of gene expression of theinterleukin IL-18, antisense mRNAs reducing or preventing thetranscription of the IL-18 mRNA or leading to degradation of the mRNA,proteins impairing correct folding, or partially or substantivelypreventing secretion of IL-18, proteases degrading the IL-18, once ithas been synthesized, and the like.

An inhibitor of IL-18 action can be an IL-18 antagonist, for example.Antagonists can either bind to or sequester the IL-18 molecule itselfwith sufficient affinity and specificity to partially or substantiallyneutralize the IL-18 or IL-18 binding site(s) responsible for IL-18binding to its ligands (like, e.g. to its receptors). An antagonist mayalso inhibit the IL-18 signaling pathway, activated within the cellsupon IL-18/receptor binding.

Inhibitors of IL-18 action may be also soluble IL-18 receptors ormolecules mimicking the receptors, or agents blocking the IL-18receptors, IL-18 antibodies, like monoclonal antibodies, for example, orany other agent or molecule preventing the binding of IL-18 to itstargets, thus diminishing or preventing triggering of the intra- orextracellular reactions mediated by IL-18.

The invention particularly relates to for the treatment or prevention ofboth acute and chronic liver diseases, such as alcoholic hepatitis,viral hepatitis, immune hepatitis, fulminant hepatitis, liver cirrhosis,and primary biliary cirrhosis, for example.

The IL-18 inhibitors contemplated herein can further be used for thetreatment of acute hepatic poisoning caused by an high amount ofparacetamol. Such an acute hepatic poisoning may be due to an overdose,be it accidental or on purpose.

As shown in the examples below, it has been surprisingly found thatIL-18 inhibitors are particularly effective in the prevention andtreatment of fulminant hepatitis (acute hepatitis). Therefore, theinvention preferably relates to the prevention and/or or treatment offulminant hepatitis.

It has been surprisingly found that a combination of an inhibitor ofIL-18 with an inhibitor of tumor necrosis factor (TNF) leads to acomplete blockade of liver injury in a murine model of disease. Theinvention therefore further relates to the use of an IL-18 inhibitor incombination with a TNF-inhibitor for the treatment and/or prevention ofliver injury.

An inhibitor of TNF can be a TNF antagonist, for example. TNFantagonists exert their activity in several ways. First, antagonists canbind to or sequester the TNF molecule itself with sufficient affinityand specificity to partially or substantially neutralize the TNF epitopeor epitopes responsible for TNF receptor binding (hereinafter termed“sequestering antagonists”). A sequestering antagonist may be, forexample, an antibody directed against TNF.

Alternatively, TNF antagonists can inhibit the TNF signaling pathwayactivated by the cell surface receptor after TNF binding (hereinaftertermed “signaling antagonists”). Both groups of antagonists are useful,either alone or together, in combination with an IL-18 inhibitor, in theprevention of therapy of liver injury.

TNF antagonists are easily identified and rated by routine screening ofcandidates for their effect on the activity of native TNF on susceptiblecell lines in vitro, for example human B cells, in which TNF causesproliferation and immunoglobulin secretion. The assay contains TNFformulation at varying dilutions of candidate antagonist, e.g. from 0.1to 100 times the molar amount of TNF used in the assay, and controlswith no TNF or only antagonist (Tucci et al., 1992).

Sequestering antagonists are the preferred TNF antagonists to be usedaccording to the present invention. Amongst sequestering antagonists,those polypeptides that bind TNF with high affinity and possess lowimmunogenicity are preferred. Soluble TNF receptor molecules andneutralizing antibodies to TNF are particularly preferred. For example,soluble TNF receptors TNF-RI and TNF-RII are useful in the presentinvention.

Truncated forms of these receptors, comprising the extracellular domainsof the receptors or functional portions thereof, are highly preferredTNF inhibitors, according to the present invention. Truncated solubleTNF type-I and type-II receptors are described in EP914431, for example.

The IL-18 inhibitor can be used simultaneously, sequentially orseparately with the TNF inhibitor.

Preferably, a combination of an IL-18 antibody or antiserum and asoluble receptor of TNF, having TNF inhibiting activity, is used.

The present invention is further based on the finding of a beneficialeffect of an IL-18 inhibitor in arthritis. The invention thereforefurther relates to the use of an IL-18 inhibitor for the manufacture ofa medicament for the treatment and/or prevention of arthritis.

According to the present invention, the term “arthritis” includes alldifferent types of arthritis and arthritic conditions, both acute andchronic arthritis, as defined for example in the Homepage of theDepartment of Orthopaedics of the University of Washington on Arthritis.Examples for arthritic conditions are ankylosing spondylitis, back pain,carpal deposition syndrome, Ehlers-Danlos-Syndrome, gout, juvenilearthritis, lupus erythematosus, myositis, osteogenesis imperfecta,osteoporosis, polyartheritis, polymyositis, psoriatic arthritis,Reiter's syndrome, scleroderma, arthritis with bowel disease, Behcets'sdisease, children's arthritis, degenerative joint disease, fibromyalgia,infectious arthritis, Lyme disease, Marfan syndrome, osteoarthritis,osteonecrosis, Pagets Disease, Polymyalgia rheumatica, pseudogout,reflex sympathetic dystrophy, rheumatoid arthritis, rheumatism,Sjogren's syndrome, familial adenomatous polyposis and the like.

Preferably, according to the invention, inhibitors of IL-18 are providedfor treatment and/or prevention of inflammatory arthritis. Inflammatoryarthritis is classified as a chronic arthritis, according to thepersistent, continuous or recurring course of the disease.

In a preferred embodiment of the invention, the inflammatory arthritisis rheumatoid arthritis (RA). RA causes inflammation in the lining ofthe joints (the synovial membrane, a one cell layer epithelium) and/orinternal organs. The disease tends to persist for many years, typicallyaffects many different joints throughout the body and ultimately cancause damage to cartilage, bone, tendons, and ligaments. The joints thatmay be affected by RA are the joints located in the neck, shoulders,elbows, hips, wrists, hands, knees, ankles and feet, for example. Inmany cases, the joints are inflamed in a symmetrical pattern in RA.

RA is prevalent in about 1% of the population in the United States,being distributed within all ethnic groups and ages. It occurs all overthe world, and women outnumber men by 3 to 1 among those having RA.

As shown in the examples below, an inhibitor of IL-18 has been proven toexhibit a highly efficacious beneficial effect on cartilage erosion. Theinvention therefore further relates to the use of an inhibitor of IL-18in the manufacture of a medicament for treatment and/or prevention ofcartilage destruction, i.e. to the use of an IL-18 inhibitor as achondroprotective agent. The IL-18 inhibitor may be used in anycondition in which cartilage destruction or erosion occurs. Cartilagedestruction is the progressive decline in the structural integrity ofjoint articular cartilage. It occurs for example in conditions affectingarticular cartilage such as rheumatoid arthritis, juvenile rheumatoidarthritis, or osteoarthritis, but also in infectious synovitis, forinstance.

In a preferred embodiment, the inhibitor of IL-18 is selected frominhibitors of caspase-1 (ICE), antibodies directed against IL-18,antibodies directed against any of the IL-18 receptor subunits,inhibitors of the IL-18 signalling pathway, antagonists of IL-18 whichcompete with IL-18 and block the IL-18 receptor, and IL-18 bindingproteins, isoforms, muteins, fused proteins, functional derivatives,active fractions or circularly permutated derivatives thereof having thesame activity.

The term “IL-18 binding proteins” is used herein synonymously with“IL18BP”. It comprises IL-18 binding proteins as defined in detailabove, including splice variants and/or isoforms of IL-18 bindingproteins. In particular, human isoforms a and c of IL-18BP are useful inaccordance with the presence invention. The proteins useful according tothe present invention may be glycosylated or non-glycosylated, they maybe derived from natural sources, like urine, or produced recombinantly.Recombinant expression may be carried out in prokaryotic expressionsystems like E. coli, or in eukaryotic, preferably in mammalian,expression systems.

The terms “muteins”, “functional derivatives”, “active fractions” and“circularly permutated derivatives” have been defined above.

Functional derivatives of IL-18BP may be conjugated to polymers in orderto improve the properties of the protein, such as the stability,half-life, bioavailability, tolerance by the human body, orimmunogenicity. To achieve this goal, IL18-BP may be linked e.g. toPolyethlyenglycol (PEG). PEGylation may be carried out by known methods,described in WO 92/13095, for example.

The term “fused protein” refers to a polypeptide comprising an IL-18BP,or a viral IL-18BP, or a mutein thereof, fused with another protein,which, e.g., has an extended residence time in body fluids. An IL-18BPor a viral IL-18BP, may thus be fused to another protein, polypeptide orthe like, e.g., an immunoglobulin or a fragment thereof. The fusion maybe direct, or via a short linker peptide which can be as short as 1 to 3amino acid residues in length or longer, for example, 13 amino acidresidues in length. Said linker may be a tripeptide of the sequenceE-F-M (Glu-Phe-Met), for example, or a 13-amino acid linker sequencecomprising Glu-Phe-Gly-Ala-Gly-Leu-Val-Leu-Gly-Gly-Gln-Phe-Met (SEQ IDNO: 14) introduced between the IL-18BP sequence and the immunoglobulinsequence. Fusion proteins comprising IL-18BP fused to all or part of animmunoglobulin are highly preferred. For example, IL-18BP may be fusedto the constant regions of Ig molecules, preferably to heavy chainregions, like the CH2 and CH3 domains of human IgG1, for example. Thegeneration of specific fusion proteins comprising IL-18BP and a portionof an immunoglobulin are described in example 11 of WP99/09063. Otherisoforms of Ig molecules are also suitable for the generation of fusionproteins according to the present invention, such as isoforms IgG₂ orIgG₄ or other Ig classes, like IgM or IgA, for example. Fusion proteinsmay be monomeric or multimeric, hetero- or homomultimeric.

In a further preferred embodiment of the invention, the inhibitor ofIL-18 is an IL-18 antibody. Anti-IL-18 antibodies may be polyclonal ormonoclonal, chimeric, fully humanised, or even fully human. Recombinantantibodies and fragments thereof are characterised by high affinitybinding to IL-18 in vivo and low toxicity. The antibodies which can beused in the invention are characterised by their ability to treatpatients for a period sufficient to have good to excellent regression oralleviation of the pathogenic condition or any symptom or group ofsymptoms related to a pathogenic condition, and a low toxicity.

Neutralising antibodies are readily raised in animals such as rabbits,goat or mice by immunisation with IL-18. Immunised mice are particularlyuseful for providing sources of B cells for the manufacture ofhybridomas, which in turn are cultured to produce large quantities ofanti-IL-18 monoclonal antibodies.

Chimeric antibodies are immunoglobulin molecules characterised by two ormore segments or portions derived from different animal species.Generally, the variable region of the chimeric antibody is derived froma non-human mammalian antibody, such as murine monoclonal antibody, andthe immunoglobulin constant region is derived from a humanimmunoglobulin molecule. Preferably, both regions and the combinationhave low immunogenicity as routinely determined (Elliott, M. J., Maini,R. N., Feldmann, M., Long-Fox, A., Charles, P., Bijl, H., and Woody, J.N., 1994). Humanised antibodies are immunoglobulin molecules created bygenetic engineering techniques in which the murine constant regions arereplaced with human counterparts while retaining the murine antigenbinding regions. The resulting mouse-human chimeric antibody preferablyhave reduced immunogenicity and improved pharmacokinetics in humans(Knight, D. M., Trinh, H., Le, J., Siegel, S., Shealy, D., McDonough,M., Scallon, B., Moore, M. A., Vilcek, J., and Daddona, P., 1993).

Thus, in a further preferred embodiment, IL-18 antibody is a humanisedIL-18 antibody. Preferred examples of humanized anti-IL-18 antibodiesare described in the European Patent Application EP 0 974 600, forexample.

In yet a further preferred embodiment, the IL-18 antibody is fullyhuman. The technology for producing human antibodies is described indetail e.g. in WO00/76310, WO99/53049, U.S. Pat. No. 6,162,963 orAU5336100. Fully human antibodies are preferably recombinant antibodies,produced in transgenic animals, e.g. xenomice, comprising all or partsof functional human Ig loci.

In a highly preferred embodiment of the present invention, the inhibitorof IL-18 is a IL-18BP, or an isoform, a mutein, fused protein,functional derivative, active fraction or circularly permutatedderivative thereof essentially having at least an activity similar toIL-18BP. Preferred active fractions have an activity which is betterthan the activity of IL-18BP or have further advantages, like a betterstability or a lower toxicity or immunogenicity, or they are easier toproduce in large quantities.

Interferons are predominantly known for inhibitory effects on viralreplication and cellular proliferation. Interferon γ, for example, playsan important role in promoting immune and inflammatory responses.Interferon β (IFN-β, an interferon type I), is said to play ananti-inflammatory role. Studies published by Triantaphyllopoulos et al.(1999) indicated that IFN-β has a beneficial effect in the therapy ofrheumatoid arthritis, as shown in a mouse model of the disease, thecollagen-induced arthritis (CIA) model. This beneficial effect of IFN-βwas confirmed in the examples below.

The invention also relates to the use of a combination of an inhibitorof IL-18 production and/or action and an interferon in the manufactureof a medicament for the treatment of arthritis, in particular rheumatoidarthritis.

Interferons may be conjugated to polymers in order to improve thestability of the proteins. A conjugate between Interferon β and thepolyol Polyethlyenglycol (PEG) has been described in WO99/55377, forinstance.

The inhibitor of IL-18 production and/or action is preferably usedsimultaneously, sequentially, or separately with the interferon.

In another preferred embodiment of the invention, the interferon isInterferon-β (IFN-β), more preferable IFN-β1a.

The invention further relates to the use of a combination of IL-18inhibitors, interferons and TNF antagonists. The combination is suitablefor the for the treatment and/or prevention of arthritis, in particularrheumatoid arthritis, and for the treatment and/or prevention of liverinjury. The active components may be used simultaneously, sequentially,or separately.

The invention further relates to the use of an expression vectorcomprising a coding sequence of an inhibitor of IL-18 in the preparationof a medicament for the prevention and/or treatment of arthriticconditions or arthritis, in particular rheumatoid arthritis, or for thetreatment of liver injury. A gene therapeutical approach is thus usedfor treating and/or preventing the disease. Advantageously, theexpression of the IL-18 inhibitor will then be in situ, thus efficientlyblocking IL-18 directly in the tissue(s) or cells affected by thedisease.

In order to treat and/or prevent arthritis, the gene therapy vectorcomprising the sequence of an inhibitor of IL-18 production and/oraction may be injected directly into the diseased joint, for example,thus avoiding problems usually involved in systemic administration ofgene therapy vectors, like dilution of the vectors, reaching of thetarget cells and side effects.

The use of a vector for inducing and/or enhancing the endogenousproduction of an inhibitor of IL-18 in a cell normally silent forexpression of an IL-18 inhibitor, or which expresses amounts of theinhibitor which are not sufficient are also contemplated according tothe invention. The vector may comprise regulatory sequences functionalin the cells desired to express the inhibitor or IL-18. Such regulatorysequences may be promoters or enhancers, for example. The regulatorysequence may then be introduced into the right locus of the genome byhomologous recombination, thus operably linking the regulatory sequencewith the gene, the expression of which is required to be induced orenhanced. The technology is usually referred to as endogenous geneactivation (EGA), and it is described e.g. in WO 91/09955. It will beunderstood by the person skilled in the art that it is also possible toshut down IL-18 expression using this technique, by introducing anegative regulation element, like e.g. a silencing element, into thegene locus of IL-18, thus leading to down-regulation or prevention ofIL-18 expression. The person skilled in the art will understand thatsuch down-regulation or silencing of IL-18 expression has the sameeffect as using an IL-18 inhibitor in order to prevent and/or treatdisease.

The invention further relates to pharmaceutical compositions forprevention and/or treatment of inflammatory arthritis or liver injury,comprising an inhibitor of IL-18. The composition may compriseantibodies against IL-18, antibodies against any of the IL-18 receptorsubunits, inhibitors of the IL-18 signalling pathway, antagonists ofIL-18 which compete with IL-18 and block the IL-18 receptor, and IL-18binding proteins, isoforms, muteins, fused proteins, functionalderivatives, active fractions or circularly permutated derivativesthereof having the same activity.

IL-18BP and its isoforms, muteins, fused proteins, functionalderivatives, active fractions or circularly permutated derivatives asdescribed above are the preferred active ingredients of thepharmaceutical compositions.

In a further preferred embodiment, the pharmaceutical compositionfurther comprises an interferon, preferably IFN-β.

In yet another preferred embodiment, the pharmaceutical compositioncomprises a TNF antagonist. Advantageously, anti-TNF antibodiesneutralising TNF action are used. Highly preferred are TBPI and TPBII asTNF antagonists.

The active ingredients of the pharmaceutical composition according tothe invention, i.e. the IL-18 inhibitor and/or interferon and/or TNFantagonist can be administered to an individual in a variety of ways.The routes of administration include intradermal, transdermal (e.g. inslow release formulations), intramuscular, intraperitoneal, intravenous,subcutaneous, oral, epidural, topical, and intranasal routes. Any othertherapeutically efficacious route of administration can be used, forexample absorption through epithelial or endothelial tissues or by genetherapy wherein a DNA molecule encoding the active agent is administeredto the patient (e.g. via a vector) which causes the active agent to beexpressed and secreted in vivo. In addition, the protein(s) according tothe invention can be administered together with other components ofbiologically active agents such as pharmaceutically acceptablesurfactants, excipients, carriers, diluents and vehicles.

In another aspect, the present invention relates to the administrationof IL-18 inhibitors to prevent or treat sepsis and is based on thefindings described in the examples. It was found in according with thepresent invention that the levels of effective (free) circulating IL-18are significantly elevated in serum of sepsis patients in comparison tohealthy individuals, and that the inhibition of IL-18 causes a decreasein IFN-γ induction by the Gram-positive bacterium Staphylococcusepidermidis.

Sepsis according to the present invention, comprises SIRS, severesepsis, septic shock, endotoxic shock etc., and may be caused byGram-positive or Gram-negative bacteria.

In addition the prevention/treatment of sepsis can be approached byadministration of an IL-18 inhibitor in combination of other cytokineinhibitors potentially able to increase its effect.

Other cytokine inhibitors can be used together with inhibitors of IL-18in the treatment of sepsis. An inhibitor of IL-18 may be used incombination with an IL-12 Inhibitor.

The term “inhibition of IL-12” within the context of this inventionrefers to any molecule modulating IL-12 production and/or action in sucha way that IL-12 production and/or action is attenuated, reduced, orpartially, substantially or completely prevented or blocked.

An inhibitor of IL-12 production can be any molecule negativelyaffecting its synthesis e.g interferon α/β (Karp et al. 2000), ormolecules negatively affecting processing or maturation of IL-12. Theinhibitors, considered according to the invention can be, for example,suppressors of gene expression of the interleukin IL-12, antisense mRNAsreducing or preventing the transcription of the IL-12 mRNA or ribozymesleading to degradation of the IL-12 mRNA, proteins impairing correctfolding, or partially or substantially preventing secretion of IL-12,proteases degrading IL-12 and the like.

IL-12 antagonists exert their activity in several ways. Antagonists canbind to or sequester the IL-12 molecule itself with sufficient affinityand specificity to partially or substantially neutralize the IL-12epitope or epitopes responsible for IL-12 receptor binding (hereinaftertermed “sequestering antagonists”). A sequestering antagonist may be,for example, an antibody directed against IL-12, a truncated form ofIL-12 receptor, comprising the extracellular domains of the receptor orfunctional portions thereof etc. An antagonist may also inhibit theIL-12 signaling pathway, which is activated within the cells uponIL-12/receptor binding (hereinafter termed “signalling antagonists”).All groups of antagonists are useful, either alone or together, incombination with an IL-18 inhibitor, in the therapy of sepsis. IL-12antagonists are easily identified and evaluated by routine screening ofcandidates for their effect on the activity of native IL-12 onsusceptible cell lines in vitro. For example mouse splenocytes in whichphorbol ester and IL-12 causes proliferation. The assay contains IL-12formulation at varying dilutions of candidate antagonist, e.g., from 0.1to 100 times the molar amount of IL-12 used in the assay, and controlswith no IL-12, antagonist only or phorbol ester and IL-2 (Tucci et al.,1992).

Sequestering antagonists are the preferred IL-12 antagonists to be usedaccording to the present invention. Amongst sequestering antagonists,antibodies that neutralize 11-12 activity are preferred. Thesimultaneous, sequential, or separate use of the IL-18 inhibitor withthe IL-12 antagonist is preferred, according to the invention. AntiIL-12 is the preferred IL-12 antagonist to be used in combination withan IL-18 inhibitor as well as antibody derivatives, fragments, regionsand biologically active portions of the antibody. The IL-18 inhibitorcan be used simultaneously, sequentially or separately with the IL-12inhibitor.

In addition, an inhibitor of IL-18 may be used in combination withinhibitors of other cytokines, known to play an important role in septicshock e.g. IL-1. TNF, IL-8 etc Dinarello 1996 and Okusawa et al. 1988.An example for an inhibitor of IL-1 is IL1-receptor antagonist, and forTNF the soluble portion of receptors TNFR1 and TNFR2.

The invention further relates to the use of an expression vectorcomprising the coding sequence of an inhibitor of IL-18 in thepreparation of a medicament for the prevention and/or treatment ofsepsis. A gene therapeutical approach is thus used for treating and/orpreventing the disease. Advantageously, the expression of the IL-18inhibitor will then be in situ, thus efficiently blocking IL-18 directlyin the tissue(s) or cells affected by the disease.

The use of a vector for inducing and/or enhancing the endogenousproduction of an inhibitor of IL-18 in a cell normally silent forexpression of an IL-18 inhibitor, or which expresses amounts of theinhibitor which are not sufficient, are also contemplated according tothe invention. The vector may comprise regulatory sequences functionalin the cells desired to express the inhibitor or IL-18. Such regulatorysequences may be promoters or enhancers, for example. The regulatorysequence may then be introduced into the right locus of the genome byhomologous recombination, thus operably linking the regulatory sequencewith the gene, the expression of which is required to be induced orenhanced. The technology is usually referred to as “endogenous geneactivation” (EGA), and it is described, e.g., in WO 91/09955.

The invention comprise also the administration of genetically modifidcells, able to produce an inhibitor of IL-18 for the treatment orprevention of sepsis.

It will be understood by the person skilled in the art that it is alsopossible to shut down IL-18 expression using the same technique, i.e.,by introducing a negative regulation element, e.g., a silencing element,into the gene locus of IL-18, thus leading to down-regulation orprevention of IL-18 expression. The person skilled in the art willunderstand that such down-regulation or silencing of IL-18 expressionhas the same effect as the use of an IL-18 inhibitor in order to preventand/or treat disease.

The definition of “pharmaceutically acceptable” is meant to encompassany carrier, which does not interfere with effectiveness of thebiological activity of the active ingredient and that is not toxic tothe host to which it is administered. For example, for parenteraladministration, the active protein(s) may be formulated in a unit dosageform for injection in vehicles such as saline, dextrose solution, serumalbumin and Ringer's solution.

For parenteral (e.g. intravenous, subcutaneous, intramuscular)administration, the active protein(s) can be formulated as a solution,suspension, emulsion or lyophilised powder in association with apharmaceutically acceptable parenteral vehicle (e.g. water, saline,dextrose solution) and additives that maintain isotonicity (e.g.mannitol) or chemical stability (e.g. preservatives and buffers). Theformulation is sterilized by commonly used techniques.

The bioavailability of the active protein(s) according to the inventioncan also be ameliorated by using conjugation procedures which increasethe half-life of the molecule in the human body, for example linking themolecule to polyethylenglycol, as described in the PCT PatentApplication WO 92/13095.

The therapeutically effective amounts of the active protein(s) will be afunction of many variables, including the type of antagonist, theaffinity of the antagonist for IL-18, any residual cytotoxic activityexhibited by the antagonists, the route of administration, the clinicalcondition of the patient (including the desirability of maintaining anon-toxic level of endogenous IL-18 activity

A “therapeutically effective amount” is such that when administered, theIL-18 inhibitor results in inhibition of the biological activity ofIL-18. The dosage administered, as single or multiple doses, to anindividual will vary depending upon a variety of factors, includingIL-18 inhibitor pharmacokinetic properties, the route of administration,patient conditions and characteristics (sex, age, body weight, health,size), extent of symptoms, concurrent treatments, frequency of treatmentand the effect desired. Adjustment and manipulation of establisheddosage ranges are well within the ability of those skilled in the art,as well as in vitro and in vivo methods of determining the inhibition ofIL-18 in an individual.

According to the invention, the inhibitor of IL-18 is used in an amountranging between about 0.1 to 5000 μg/kg of body weight or about 1 to1000 μg/kg of body weight. Amounts of about 10 to 500 μg/kg of bodyweight are preferred, and amounts of about 50 to 250 μg/kg of bodyweight are further preferred.

Inhibitors of IL-18 may also be used in an amount of about 1 to 50 μg/kgof body weight.

The route of administration which is preferred according to theinvention is administration by subcutaneous route. Intramuscularadministration is further preferred according to the invention.

In further preferred embodiments, the inhibitor of IL-18 is administereddaily or every other day.

The daily doses are usually given in divided doses or in sustainedrelease form effective to obtain the desired results. Second orsubsequent administrations can be performed at a dosage which is thesame, less than or greater than the initial or previous doseadministered to the individual. A second or subsequent administrationcan be administered during or prior to onset of the disease.

According to the invention, the IL-18 inhibitor can be administeredprophylactically or therapeutically to an individual prior to,simultaneously or sequentially with other therapeutic regimens or agents(e.g. multiple drug regimens), in a therapeutically effective amount, inparticular with an interferon and/or a TNF antagonist. Active agentsthat are administered simultaneously with other therapeutic agents canbe administered in the same or different compositions. Other agentswhich may be used in combination with IL-18 inhibitors, in particularwith IL-18BP, are COX-2 inhibitors. COX-2 inhibitors are know in theart. Specific COX-2 inhibitors are disclosed in WO 01/00229, forexample.

The invention further relates to a method for the preparation of apharmaceutical composition comprising admixing an effective amount of anIL-18 inhibitor with a pharmaceutically acceptable carrier.

Having now described the invention, it will be more readily understoodby reference to the following examples that are provided by way ofillustration and are not intended to be limiting of the presentinvention.

EXAMPLES PART I: Examples 1 to 17 Isolation, Production and BiologicalActivities of IL-18BP Example 1: Isolation of an IL-18 Binding Protein

E. coli IL-18 (2.5 mg, Peprotech, N.J.) was coupled to Affigel-10 (0.5ml, BioRad), according to the manufacturer's instructions and packedinto a column. Crude urinary proteins (1000-fold concentrated, 500 ml)were loaded onto the column at a flow rate of 0.25 ml/min. The columnwas washed with 250 ml 0.5M NaCl in phosphate buffered saline (PBS).Bound proteins were then eluted with 25 mM citric acid, pH 2.2 andbenzamidine (1 mM), immediately neutralized by 1M Na₂CO₃. Fractions of 1ml were collected. The fractions were analyzed by SDS-PAGE and silverstaining. The IL-18 binding protein eluted in fractions 2-8 as a ˜40,000Dalton protein (FIG. 1). The ˜40 kD band, corresponding to the IL-18BPexhibited a distinct yellow color upon silver staining. The variousfractions were analyzed by cross-linking with ¹²⁵I-IL-18, SDS-PAGE andautoradiography as described in Example 2. An IL-18 binding protein wasfound in fractions 2-8, eluted from the IL-18-agarose column (FIG. 2).

Example 2: Cross-Linking of Affinity-Purified IL-18Bp to Labeled IL-18

Samples (40 μl) of IL-18BP from the affinity purification step wereincubated (70 min. at 4° C.) with ¹²⁵I-IL-18 (5,000,000 cpm).Disuccinimidyl suberate (DSS), dissolved in dimethyl sulfoxide (Me₂SO,20 mM), was then added to a final concentration of 2 mM and the mixturewas left for 20 min. at 4° C. The reaction was stopped by the additionof 1M Tris-HCl pH 7.5, and 1M NaCl to a final concentration of 100 mM. Asample buffer containing Dithiothreitol (DTT, 25 mM final) was added andthe mixtures were analyzed by SDS-PAGE (7.5% acrylamide) followed byautoradiography (FIG. 2).

A specific band of molecular weight 58 kD, probably consisting of a ˜40kD protein cross-linked to the ˜20 kD ¹²⁵I-IL-18, was observed infractions eluted from the IL-18 affinity column (lanes 2 and 3) but notin the column wash (lane 1), containing all other crude urinaryproteins.

Example 3: Protein Sequence Analysis

Eluted fractions from the affinity column of Example 1 were resolved bySDS-PAGE (10% acrylamide) under non-reducing conditions, electroblottedon a PVDF membrane (Pro-Blot, Applied Biosystems, USA). The membrane wasstained with coomassie blue, the ˜40 kD band was excised and subjectedto protein sequence analysis by a Procise microsequencer (AppliedBiosystems, USA). The following major sequence was obtained:

T-P-V-S-Q-Q-x-x-x-A-A-A (SEQ ID NO: 11) 1 . . . 5 . . . .10 . .

wherein x represents a yet undetermined amino acid.

-   -   In addition, a minor sequence was obtained:

A-x-Y-x-R-I-P-A-x-A-I-A (SEQ ID NO: 15) 1 . . . 5 . . . .10 . .

Because of this double sequence it was not possible to obtain a longersequence data. The minor sequence was identified as a fragment of humandefensin, (accession No. p11398) starting at amino acid 65 of defensin.The major sequence could not be associated with any other known protein,as determined by searching all available databases in NCBI and TIGR bythe blastp and tblastn search programs.

In order to obtain a longer and more accurate sequence and in order toidentify potential cysteine residues, another aliquot of the fractioneluted from the IL-18-agarose column was reduced with DTT in 6 Mguanidine HCl, reacted with 4-vinyl pyridine, desalted by amicro-ultrafiltration device (Ultrafree, cutoff 10,000 Daltons,Millipore) and subjected to protein microsequence analysis. After cycleNo. 1 of sequencing, the filter was reacted with o-phtalaldehyde toblock all N-terminal polypeptides other than Pro. In this way only themajor sequence was obtained as follows:

(SEQ ID NO: 10) TPVSQXXXAA XASVRSTKDP CPSQPPVFPA AKQCPALEVT1       10         20         30         40 (T = Thr; P = Pro; V = Val;S = Ser; Q = Gln; X = Unknown; A = Ala; R = Arg; K = Lys; D = Asp; C =Cys; F = Phe; L = Leu; E = Glu)

In cycles 6,7,8 and 11 a low level of a Thr signal was obtained. Becauseof this low level we considered it more prudent not to assign a specificamino acid residue at said cycles.

The resulting sequence is significantly different from that of any otherknown protein, as determined by searching protein databases. However,searching the TIGR database by the tblastn search program provided acDNA file, denoted THC123801, whose open reading frame (218 codons),when translated contains a sequence highly homologous to that of theN-terminal sequence of IL-18BP. The homology is hereby shown:

(The upper sequence (1-40) (SEQ ID NO: 12) is that of the IL-18BPisolated according to the invention; the lower sequence (51-100) (SEQ IDNO: 13) is deduced by translation of the cDNA of TIGR file THC123801).

The putative protein sequence, obtained by translating file THC123801,was ambiguous at residues 2 and 4 of the IL-18BP. It confirms theidentity of amino acid residues 6, 7 and 8 of IL-18BP as Thr and seemsto do so also for residue 11.

Example 4: The IL-18BP is a Glycoprotein

Aliquot (0.3 ml) of eluted fractions of Example 1 were further purifiedby size exclusion chromatography on a Superose 12 column (1×30 cm,Pharmacia, Sweden). The column was pre-equilibrated and eluted withphosphate buffered saline and sodium azide (0.02%) at a flow rate of 0.5ml/min. and fractions of 1 min. were collected. The IL-18 bindingprotein eluted in fractions 20-25 as a ˜40,000 Dalton protein, asdetermined by SDS-PAGE and silver staining. A sample containing the˜40,000 Dalton protein (fraction 23, 50 μl, ˜50 ng protein) was reactedwith N-glycosidase F (PNGase F, Biolab) according to the manufacturersinstructions. Briefly, the aliquot was denatured by boiling in thepresence of 5% SDS for 10 min., 10×G7 buffer (2.5 ?l), 10% NP-40 (2.5μl) and PNGase F (1 μl), 1 h at 37° C. The sample was analyzed bySDS-PAGE (10% acrylamide) under non-reducing conditions and comparedwith undigested IL-18BP from the same Superose 12 fraction. It was foundthat the ˜40 kD band of IL-18BP disappeared in the PNGase-treatedfraction. New bands, corresponding to 30 kD (just above the PNGase band)and 20 kD were obtained. The elimination of the ˜40 kD band indicatesthat this band is an N-glycosylated protein.

Example 5: Blocking of the Biological Activity of IL-18 by IL-18BP

The ability of the IL-18BP isolated from urine to block IL-18 activitywas determined by measuring the IL-18-induced production of IFN-γ inmononuclear cells. IL-18 induces IFN-γ when added together with eitherlow concentration of LPS, IL-12, IL-2, or other stimulants. The activityof IL-18 was tested in murine splenocytes, in human peripheral bloodmononuclear cells (PBMC) and in the human KG-1 cell line. Spleen cellswere prepared from a healthy mouse, washed and suspended in RPMI 1640medium supplemented with 10% fetal bovine serum at 5×10⁶ cells/ml. 1.0ml cultures were stimulated with LPS (either 0.5 or 1 μg/ml) togetherwith recombinant human or murine IL-18 (either 0.5 or 5 ng/ml). HumanIL-18 binding protein (0.5 or 50 ng/ml) was added to the recombinantIL-18 before adding to spleen cells. After culturing for 24 h, thespleen cells were subjected to three freeze (−70° C.) and thaw (roomtemperature) cycles, the cellular debris was removed by centrifugationand the supernatants were assayed for IFN-γ using the ELISA kits formouse IFN-γ (Endogen). As shown in FIG. 3A, IL-18BP blocked the activityof huIL-18 in murine splenocytes in a dose-dependent manner. Incontrast, as a control, soluble interferon-α/6 receptor had no effect.The activity of recombinant murine IL-18 was similarly inhibited by thehuman IL-18BP, suggesting that human IL-18BP recognizes murine IL-18(FIG. 3B). Endogenous IL-18 is induced in murine splenocytes by highconcentrations of LPS, leading to production of IFN-γ. Indeed, IFN-γinduction by LPS (10 μg/ml) was also inhibited by the urinary IL-18BP(FIG. 3C). Concanavalin A (con A) activates T-cells to produce IFN-γ inthe absence of IL-18 (Fantuzzi et al., 1998). Indeed, induction of IFN-γby Con A was not inhibited by IL-18BP even at high concentrations (FIG.3D). This observation demonstrated that IL-18BP was a specific inhibitorof IL-18 bioactivity rather than a non-specific inhibitor of IFN-γproduction. IL-18BP also inhibited the activity of human IL-18 in humanKG-1 cells induced by a combination of IL-18 and TNF-α (FIG. 3E).

The above data demonstrate that urinary IL-18BP inhibits human as wellas murine IL-18 activity as measured by co-induction of IFN-γ in humanand murine mononuclear cells. The concentration of IL-18BP which reducedIL-18 activity by >90% was comparable to that of IL-18 itself,suggesting a high affinity interaction between these two proteins.

Example 6: Isolation of cDNA Clones Coding for IL-18BP

Total RNA from Jurkat T-cells (CRL 8163, American Type CultureCollection) was reverse-transcribed with SuperScript RNase H⁻ reversetranscriptase (Gibco-BRL) and random primers (Promega, Madison Wis.).The resulting cDNA fragments were then amplified by PCR, using Taq DNApolymerase (Sigma) and primers corresponding to TIGR clone THC123801nucleotides 24-44 (sense) and 500-481 (reverse). The amplification wasdone in 30 cycles of annealing (55° C., 2 min) and extension (70° C., 1min). The resulting PCR products were resolved by agarose (1%) gelelectrophoresis, eluted and cloned into pGEM-Teasy TA cloning vector(Promega). DNA from individual clones was sequenced with T7 and SP6primers.

The resulting 477 by fragment was ³²P-labeled by random priming. Thisprobe was used for screening various human cDNA and genomic libraries.Duplicate nitrocellulose filters were lifted and hybridized with theprobe at 60° C. in a buffer consisting of 6×SSC, 10×Denhardt's solution,0.1% SDS and 100 μg/ml Salmon sperm DNA. The filters were washed andexposed overnight at −80° C. to Kodak XAR film. Double positive cloneswere plaque-purified. Plasmids were excised from the XpCEV9 clones andself-ligated. cDNA clones from other libraries were isolated accordingto the manufacturer's instructions. Automated DNA sequence analysis ofthe isolated clones was performed with Models 373A and 377 sequencers(Applied Biosystems) using sense and antisense primers. Standardprotocols were used for these cloning procedures (Sambrook et al.,1989).

The following libraries were screened: a human monocyte cDNA library,constructed in λpCEV9 cloning vector (Gutkind, 1991), kindly provided byT. Miki; a human Jurkat leukemic T-cell cDNA library, a human peripheralblood leukocyte cDNA library and a human spleen cDNA library, all fromClontech (Palo Alto, Calif.). A human placenta genomic library in lambdaFIX II vector was from Stratagene (La Jolla, Calif.).

All cDNA clones corresponded to four different IL-18BP splice variantswere obtained and characterized. All splice variants coded for putativesoluble secreted proteins. The most abundant one (IL-18BPa) had an openreading frame of 192 codons, coding for a signal peptide of 28 aminoacid residues followed by a mature putative IL-18BPa, whose first 40residues (SEQ ID NO:10) matched perfectly with the N-terminal proteinsequence of the urinary IL-18BP (SEQ ID NO:2). The position of thecysteine residues suggested that this polypeptide belongs to theimmunoglobulin (Ig) super-family. Each of the four Gln residues withinmature IL-18BPa was a potential N-glycosylation site. The other threesplice variants of IL-18BP were significantly less abundant.

Another 1 kb IL-18BPb cDNA coded for a mature protein of 85 amino acidresidues (SEQ ID NO:4). A third variant, IL-18BPc, was represented by a2.3 kb cDNA, coding for a mature IL-18BP of 169 amino acid residues (SEQID NO:6). The fourth variant, IL-18BPd coded for a mature IL-18BP of 133amino acid residues (SEQ ID NO:8). In-exon splicing occurred at twosites along the pro-mRNA. These events and an additional 5′ exon inIL-18BPd gave rise to 3 different 5′ UTRs in the various cDNA clones. Itis therefore quite possible that different IL-18BP variants may begenerated in response to distinct transcription regulation signals.

No cDNA coding for a receptor with a transmembrane domain was found sofar.

Example 7: Construction of a Mammalian Expression Vector, Production ofRecombinant IL-18Bp, and Evaluation of the Biological Activities ofRecombinant IL-18BP

The coding region of the IL-18BPa cDNA was amplified by PCR with thesense primer

(SEQ ID NO: 16) 5′ TATATCTAGAGCCACCATGAGACACAACTGGACACCA

and the reverse primer:

(SEQ ID NO: 17) 5′ ATATCTAGATTAATGATGATGATGATGATGACCCTGCTGCTGTGGAC TGC

The PCR products were cut with Xba I and cloned into the Xba I site ofthe pEF-BOS expression vector (25), to yield pEF-BOS-IL-18BPa. Theconstructs were confirmed by DNA sequencing.

Batches of 6×10⁷ COS7 cells in 1.4 ml TD buffer, containingpEF-BOS-IL-18BPa plasmid DNA (10 μg) and DEAE-dextran (120 μg) wereincubated for 30 min at room temperature, as described (Sampayrat andDana, 1981). The cells were then washed with DMEM-10% FBS, plated for 4hr in DMEM-10, washed and incubated for 3-5 days in serum-free DMEM.Culture medium was collected, concentrated 6-fold by ultrafiltration (10kD cutoff) and the IL-18BP-His₆ was isolated on a Talon column(Clontech) with imidazole as eluant according to the manufacturer'sinstructions.

Immunological cross-reactivity of the urinary and the COS7-expressedIL-18BP was assessed as follows: Urinary IL-18BP (5 μg) was labeled with¹²⁵I by the chloramine T procedure. Supernatants of COS7 cells (250 μl)were mixed (1 h, room temperature final volume 500 μl) with the antibodyto urinary IL-18BP, diluted 1:1000 in phosphate-buffered saline (PBS),0.05% Tween 20 and 0.5% bovine serum albumin (Wash Buffer). ¹²⁵I-labeledurinary IL-18BP (10⁶ cpm) was then added and after 1 h proteinG-sepharose (20 μl) was added. The mixture was suspended (1.5 h, 4° C.),the beads were then isolated and washed wash 3× Wash Buffer and once inPBS. The beads were then eluted with a Sample buffer, resolved bySDS-PAGE (10% acrylamide under reducing conditions followed byAutoradiography.

IL-18BPa ran as a single band upon SDS-PAGE with silver staining underreducing and non-reducing conditions and had the same apparent molecularmass as that of the urinary IL-18BP (data not shown). Protein sequenceanalysis of this preparation revealed the same N-terminal sequence asthat of the urinary IL-18BP, indicating that the latter was not degradedat its N-terminus.

Immunoblot analysis of IL-18BPa with antibodies raised against theurinary IL-18BP revealed the same molecular mass band as that of theurinary protein. Furthermore, using immunoprecipitation followed bySDS-PAGE and autoradiography, IL-18BPa was able to displace urinary¹²⁵I-IL-18BP from binding to the antibody. Therefore, IL-18BPacorresponds structurally to the urinary IL-18BP.

Crude and purified IL-18BPa was tested for its ability to inhibit thebiological activity of IL-18. IL-18BPa inhibited in a dose dependentmanner the IFN-γ inducing activity of human and mouse IL-18 in murinesplenocytes, PBMC and the human KG-1 cell line (FIG. 4).

The results of the various bioassays as well as the mobility shift assay(Example 8) demonstrated that inhibition of IL-18 activity is anintrinsic property of the cloned IL-18BP and not that of anyaccompanying impurities in urinary IL-18BP, such as the co-elutingfragment of defensin.

Example 8: Electrophoretic Mobility Shift Assays

The effect of the urinary and the recombinant IL-18BP on IL-18-inducedactivation of NF-κB in human KG-1 cells was also studied. Human KG-1cells (4×10⁶ in 1 ml RMPI) were stimulated with either huIL-18 (10ng/ml) or huIL-18 pre-mixed with an IL-18BP (20 min, room temperature).After 20 min at 37° C., cells were washed three times with ice-cold PBSand immediately frozen in liquid nitrogen. Cell pellets were resuspendedin three times the packed cell volume in buffer A (20 mM Tris pH 7.6,0.4M NaCl, 0.2 mM EDTA, glycerol (20% by volume), 1.5 mM MgCl₂, 2 mMdithiothreitol (DDT), 0.4 mM PMSF, 1 mM Na₃VO₄, 2 μg/ml each ofleupeptin, pepstatin and aprotinin). Cell debris was removed bycentrifugation (15,000×g, 15 min.), aliquots of the supernatant werefrozen in liquid nitrogen and stored at −80° C. Protein concentrationwas determined by a Bradford assay (Bio-Rad) using bovine serum albuminas standard. A double-stranded oligonucleotide corresponding to NF-κBbinding element (10 pmol, Promega) was labeled with [³²P]dCTP (300Ci/mmol) and T4 polynucleotide kinase (New England Biolabs). Freenucleotides were removed by a spin column. Extracts (10 μg protein) ofcells treated with IL-18 or IL-18 plus IL-18BP were incubated (15 min.,room temperature) with the labeled probe (3×10⁴ cpm) together with polydl.dC (500 ng, Pharmacia) and denatured salmon sperm DNA (100 ng, Sigma)in 20 μl buffer consisting of HEPES (pH 7.5, 10 mM), 60 mM KCl, 1 mMMgCl₂, 2 mM EDTA, 1 mM DTT and glycerol (5% by volume). The mixtureswere then loaded onto 5% non-denaturing polyacrylamide gels.Electrophoresis was performed at 185 V in 0.5×TBE (40 mM Tris HCl, 45 mMboric acid and 2.5 mM EDTA). Gels were vacuum dried and autoradiographedovernight at −80° C. IL-18 was found to induce the formation of the p50NF-?B homodimer and the p65/p50 NF-κB heterodimer. Urinary as well asrecombinant IL-18BP inhibited the activation of NF-κB by IL-18, asdetermined by an electrophoretic mobility shift assay with KG-1 cellextracts binding a radiolabeled oligonucleotide corresponding to theNF-κB consensus sequence.

Example 9: Expression of IL-18BP in E. coli, Yeast and Insect Cells

IL-18BP may also be produced by other recombinant cells such asprokaryotic cells, e.g., E. coli, or other eukaryotic cells, such asyeast and insect cells. Well known methods are available forconstructing appropriate vectors, carrying DNA that codes for eitherIL-18BP and suitable for transforming E. coli and yeast cells, orinfecting insect cells in order to produce recombinant IL-18BP. Forexpression in yeast cells, the DNA coding for IL-18BP (Example 6) is cutout and inserted into expression vectors suitable for transfection ofyeast cells. For expression in insect cells, a DNA coding for IL-18BP isinserted into baculovirus and the insect cells are infected with saidrecombinant baculovirus. For expression in E. coli, the DNA coding forIL-18BP is subjected to site directed mutagenesis with appropriateoligonucleotides, so that an initiation ATG codon is inserted just priorto the first codon of mature IL-18BP. Alternatively, such DNA can beprepared by PCR with suitable sense and antisense primers. The resultingcDNA constructs are then inserted into appropriately constructedprokaryotic expression vectors by techniques well known in the art(Manatis, 1982).

Example 10: Construction of Adeno-Associated Expression Vector for InVivo Expression of IL-18BPa

A functional gene coding for IL-18BPa is constructed based on plasmidpcDNA3 (Invitrogen, San Diego Calif.). The IL-18BP cDNA with a Kozakconsensus sequence at the 5′ end is ligated into the Xba I site ofpcDNA3 in a way that destroys the restriction site. New Xba I sites areinserted by site-directed mutagenesis before the neomycin cassette (base2151 of the original pcDNA3 sequence) and after the SV40 polyadenylationsignal (base 3372 of the original pcDNA3 sequence). This construct isthen cut with Xba I and the resulting 4.7 kb minigen is inserted at theXba I site of plasmid psub201 as described (Snyder et al, 1996, CurrentProtocols in Human Genetics, Chapters 12.1.1-12.1.17, John Wiley &Sons). The resulting recombinant plasmid is cotransfected with thehelper AAV plasmid pAAV/Ad into human T293 cells. The cultures are theninfected with adenovirus as a helper virus and the cells are collectedafter 48-60 hours of incubation. The cells are subjected to 3freeze-thaw cycles, cell debris is removed by centrifugation, and thesupernatant is brought to 33% saturation with ammonium sulfate. Themixture is then centrifuged and rAAV is precipitated from thesupernatant by bringing the ammonium sulfate to 50% saturation. Thevirus is further purified by CsCl, dialyzed and finally heated for 15min at 56° C. to destroy any adenovirus.

Example 11: Construction of Recombinant Fusion Proteins of IL-18BP

The production of proteins comprising IL-18BP fused to the constantregion of IgG2 heavy chain may be carried out as follows: the DNA ofIL-18BP is subjected to site-directed mutagenesis with appropriateoligonucleotides so that a unique restriction site is introducedimmediately before and after the coding sequences. A plasmid bearing theconstant region of IgG2 heavy chain, e.g. pRKCO42Fc1 (Byrn, 1990) issubjected to similar site-directed mutagenesis to introduce the sameunique restriction site as close as possible to Asp 216 of IgG1 heavychain in a way that allows translation in phase of the fused protein. AdsDNA fragment, consisting of 5′ non-translated sequences and encodingfor IL-18BP is prepared by digestion at the unique restriction sites oralternatively by PCR with appropriately designed primers. The mutatedpRKCD42Fc1 is similarly digested to generate a large fragment containingthe plasmid and the IgG1 sequences. The two fragments are then ligatedto generate a new plasmid, encoding a polypeptide precursor consistingof IL-18BP and about 227 C-terminal amino acids of IgG1 heavy chain(hinge region and CH2 and CH3 domains). The DNA encoding the fusedproteins may be isolated from the plasmid by digestion with appropriaterestriction enzymes and then inserted into efficient prokaryotic oreukaryotic expression vectors.

Example 12: Production of Chemically Modified IL-18BPs

In order to increase the half-life of the IL-18BPs in plasma, IL-18BPswhich are chemically modified with polyethylene glycol (PEG) may bemade. The modification may be done by cross linking PEG to a cysteineresidue of the IL-18BP molecules. Mutant IL-18BPs may be constructedwhich contain an extra cysteine residue at the amino terminus,glycosylation sites, and the carboxyl terminus of each IL-18BP. Themutagenesis may be carried out by PCR using oligonucleotides containingthe desired mutation. These mutant proteins are expressed in the usualmanner as well known in the art. Pegylation of these proteins will becarried out and the activity will be assessed.

Example 13: Preparation of Polyclonal Antibodies to IL-18BP

Rabbits were initially injected subcutaneously with 5 μg of a purepreparation of the urinary IL-18BP, emulsified in complete Freund'sadjuvant. Three weeks later, they were injected again subcutaneouslywith 5 μg of the IL-18BP preparation in incomplete Freund's adjuvant.Two additional injections of IL-18BP as solution in PBS were given at 10day intervals. The rabbits were bled 10 days after the lastimmunization. The development of antibody level was followed by aradioimmunoassay. ¹²⁵I-labeled IL-18BP (166,000 cpm) was mixed withvarious dilutions (1:50, 1:500, 1:5,000 and 1:50,000) of the rabbitserum. A suspension of protein-G agarose beads (20 μl, Pharmacia) wasadded in a total volume of 200 μl. The mixture was left for 1 hour atroom temperature, the beads were then washed 3 times and boundradioactivity was counted. Rabbit antiserum to human leptin was used asa negative control. The titer of the IL-18R antiserum was between 1:500and 1:5000, while that of the negative control was less than 1:50.

Example 14: Preparation of Monoclonal Antibodies to IL-18BP

Female Balb/C mice (3 months old) are first injected with 2 μg purifiedIL-18BP in an emulsion of complete Freund's adjuvant, and three weekslater, subcutaneously in incomplete Freund's adjuvant. Three additionalinjections are given at 10 day intervals, subcutaneously in PBS. Finalboosts are given intraperitoneally 4 and 3 days before the fusion to themouse showing the highest binding titer as determined by IRIA (seebelow). Fusion is performed using NSO/1 myeloma cell line andlymphocytes prepared from both the spleen and lymph nodes of the animalas fusion partners. The fused cells are distributed into microcultureplates and the hybridomas are selected in DMEM supplemented with HAT and15% horse serum. Hybridomas that are found to produce antibodies toIL-18BP are subcloned by the limiting dilution method and injected intoBalb/C mice that had been primed with pristane for the production ofascites. The isotypes of the antibodies are defined with the use of acommercially available ELISA kit (Amersham, UK).

The screening of hybridomas producing anti-IL-18BP monoclonal antibodiesis performed as follows: Hybridoma supernatants are tested for thepresence of anti-IL-18BP antibodies by an inverted solid phaseradioimmunoassay (IRIA). ELISA plates (Dynatech Laboratories,Alexandria, Va.) are coated with Talon-purified IL-18BPa-His₆ (10 μg/ml,100 μl/well). Following overnight incubation at 4° C., the plates arewashed twice with PBS containing BSA (0.5%) and Tween 20 (0.05%) andblocked in washing solution for at least 2 hrs at 37° C. Hybridomaculture supernatants (100 Owen) are added and the plates are incubatedfor 4 hrs at 37° C. The plates are washed 3 times and a conjugate ofgoat-anti-mouse horseradish peroxidase (HRP, Jackson Labs, 1:10,000, 100Owen) is added for 2 hrs at room temperature. The plates are washed 4times and the color is developed by ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid, Sigma) with H₂O₂ as a substrate.The plates are read by an automatic ELISA reader. Samples giving OD thatare at least 5 times higher than the negative control value areconsidered positive.

Example 15: Affinity Chromatography of IL-18BP with MonoclonalAntibodies

Antibodies against IL-18BP are utilized for the purification of IL-18BPby affinity chromatography. Ascitic fluid containing the monoclonalantibody secreted by the hybridoma is purified by ammonium sulfateprecipitation at 50% saturation followed by extensive dialysis againstPBS. About 10 mg of immunoglobulins are bound to 1 ml Affigel 10 (BioRadUSA), as specified by the manufacturer.

250 ml of human urinary proteins (equivalent to 250 l of crude urine)are loaded on 0.5 ml of the anti IL-18BP antibody column at 4° C. at aflow rate of 0.25 ml/min. The column is washed with PBS until no proteinis detected in the washings. IL-18BP is eluted by 25 mM citric acidbuffer, pH 2.2 (8×1 column volume fractions) and immediately neutralizedby 1 M Na₂CO₃. Further purification of this preparation is obtained bysize exclusion chromatography.

Example 16: ELISA Test

Microtiter plates (Dynatech or Maxisorb, by Nunc) are coated withanti-IL-18BP monoclonal antibody (serum free hybridoma supernatant orascitic fluid immunoglobulins) overnight at 4° C. The plates are washedwith PBS containing BSA (0.5%) and Tween 20 (0.05%) and blocked in thesame solution for at least 2 hrs at 37° C. The tested samples arediluted in the blocking solution and added to the wells (100 μl/well)for 4 hrs at 37° C. The plates are then washed 3 times with PBScontaining Tween 20 (0.05%) followed by the addition of rabbitanti-IL-18BP serum (1:1000, 100 μl/well) for further incubationovernight at 4° C. The plates are washed 3 times and a conjugate ofgoat-anti-rabbit horseradish peroxidase (HRP, Jackson Labs, 1:10,000,100 Owen) was added for 2 hrs at room temperature. The plates werewashed 4 times and the color is developed by ABTS(2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid, Sigma) with H₂O₂as a substrate. The plates are read by an automatic ELISA reader.

Example 17: Non-Glycosylated Human IL-18BP is Biologically Active

Purified recombinant IL-18BPa was tested for its ability to inhibit thebiological activity of IL-18. IL-18BPa inhibited in a dose dependentmanner the IFN-γ inducing activity of human and mouse IL-18 in murinesplenocytes, PBMC and the human KG-1 cell line (FIG. 4).

Purified IL-18BPa having an His₆ tag at the C-terminus (1.5 μg, 50 μl)was adjusted to pH 7.5 and mixed with N-glycosidase F (3 μl, 500,000U/ml, PNGase F, New England Biolabs). The mixture was incubated for 24 hat 37° C. under non-denaturing conditions. Aliquots from the sample andfrom undigested IL-18BP-His₆ were analyzed by SDS-PAGE undernon-reducing conditions followed by immunoblotting with antibodies toIL-18PB. It was found that the ˜40 kD band of IL-18BP-His₆ disappearedin the PNGase-treated fraction and a new ˜20 kD band was obtained. Themolecular mass of the product and the specificity of PNGase F indicatedthat IL-18BP-His₆ was fully deglycosylated.

The PNGase-treated fraction, undigested IL-18BP-His₆ and control samplecontaining PNGase in buffer were absorbed separately on Talon beads,washed with phosphate buffer and eluted with imidazole (100 mM). Theeluted fractions were subjected to bioassay using human IL-18 (20ng/ml), LPS (2 μg/ml) and murine splenocytes. The results are shown inthe following Table 4:

TABLE 4 Sample IFN-? (ng/ml) Control 7.5 Non-digested IL-18BP-His₆ 0PNGase-treated IL-18BP-His₆ 0

Therefore, it is concluded that deglycosylated IL-18BP is biologicallyactive as a modulator of IL-18 activity.

PART II: Examples 18 to 25 Relating to the Use of IL-18 Inhibitors inLiver Injury Example 18: Production of IL-18BP-His Tag

Purified recombinant human IL18BP containing a his-tag (r-hIL-18BP-Histag) was produced in CHO cells. The production of recombinant proteinsin eukaryotic cells is known by the person skilled in the art. Wellknown methods are available for constructing appropriate vectors,carrying DNA that codes for IL-18BP and suitable for transfection ofeukaryotic cells in order to produce recombinant IL-18BP. For expressionin cells, the DNA coding for IL-18BP (see, e.g. (Novick et al., 1999) iscut out and inserted into expression vectors suitable for transfectionof cells. Alternatively, such DNA can be prepared by PCR with suitablesense and antisense primers. The resulting cDNA constructs are theninserted into appropriately constructed eukaryotic expression vectors bytechniques well known in the art (Manaitis, 1982). The recombinantprotein was purified to over 95% purity and found to be biologicallyactive in-vitro and in-vivo with a high affinity to its ligand.

Example 19: Protective Effect of IL18BP Against Endotoxin-Induced Deathin the Murine Model

A murine model was used to test whether IL18BP, an inhibitor of IL-18,would protect mice against a high dose of lipopolysaccharides (LPS). LPSelicits acute liver injury, followed by rapid death of the mice.

4 mg/kg of recombinant, human IL-18BP (rhIL18BPhis) containing a his-tag(resulting from recombinant production of the protein) was injectedintraperitoneally (i.p). into C57BL/6 mice. 1 h later, 60 mg/kg LPS wereinjected (lethal doses). The survival of mice was compared to a group ofanimals who received LPS alone (no IL18BP).

Five out of 7 mice injected with rhIL-18BP-his survived the LPSinjection in contrast to the control mice, in which all animals diedwithin 3 days.

Blood samples were taken 5 h after the LPS injection in the absence orpresence of increasing doses of rhIL-18BP-his and analyzed by ELISA forcirculating IFN-γ (FIG. 5). 0.4 and 4 mg/kg rhIL-18BP induced a 2 foldreduction in serum IFN-γ. This inhibition was lost at lower doses ofrhIL-18BP (0.004 and 0.4 mg/kg).

Example 20: IL18BP has a Protective Effect Against Liver Injury in aMurine Model

A mouse model of fulminant hepatitis was used to test the effect ofIL18BP. Mice develop acute liver injury when subjected to a sequentialadministration of Propionibacterium acnes (P. acnes) andlipopolysaccharide (LPS).

Mice were injected with increasing doses of rhIL-18BP-his (4; 0.4; 0.04;0 mg/kg) at various times (1 h, 20 min, simultaneously) before theinjection of LPS in C57BL/6 P. acnes sensitized mice. When rhIL-18BP-hiswas given i.p. at the same time as LPS, none of the mice survived andlevels of circulating IFN-γ and TNF-α were unaffected. Surprisingly,rhIL-18BP (4 and 0.4 mg/kg) induced a 70% reduction of circulatingAlanine aminotransferase (a marker of liver injury), as shown in FIG. 6.

In addition to this, the survival of mice was monitored (FIG. 7): WhenrhIL-18BP was given i.p. 20 minutes before LPS, the two highest doses ofII-18BP (4 and 0.4 mg/kg) delayed the death of the mice by 10 h ascompared to the control mice who received NaCl instead of IL-18BP

The results of the measurement of serum IFN-γ levels are shown in FIG.8. rhIL-18BP (4 mg/kg) inhibited 90% of circulating IFN-γ levels and 80%of circulating Alanine aminotransferase (not shown).

When rhIL-18BP-his was given 1 h before LPS, survival curves and levelsof circulating IFN-γ were similar to what was observed whenrhIL-18BP-his was given 20 min before LPS, but levels of circulatingalanine aminotransferase were unaffected (not shown).

In addition to this, murine liver tissue was analyzed byhematoxilin-eosine staining, as well as by tunnel microscopy. The liversof mice, in which severe hepatitis had been induced before, showedsevere necrosis as compared to normal liver tissue. In contrast to this,liver tissue of mice treated with IL-18BP showed significantly lessnecrotic foci than untreated mice.

Example 21: Anti-IL-18 Antibodies Protect Against Lethal Endotoxemia

In order to evaluate, whether blockade of IL-18 with IL-18 antibodieswould protect mice against lethal doses of bacteriallipopolysaccharides, C57BL/6J mice were first injected with aneutralizing rabbit anti-mouse IL-18 antibody (polyclonal) or normalrabbit serum (NDS) as a control. 30 min after antibody treatment, alethal dosis of LPS derived either from E. coli (FIG. 9 A) or S.thyphimurium (FIG. 9 B) was injected. Experiments involved 10-12mice/group, and were performed twice on two different occasions.

As shown in FIG. 9A, treatment of the mice with the anti-IL-18 antiserumprevented the mortality induced by 40 mg/kg E. coli LPS. 100% of themice survived after anti-IL-18 treatment vs. 10% survival in micetreated with normal rabbit serum (p<0.005).

FIG. 9B shows that the antibody treated mice were also protected againstS. typhimurium lethal effects (50% vs. 0% survival; p<0.05).

Example 22: Blockade of IL-18 and TNF-α Protects Mice from ConA- andPEA-Induced Hepatotoxicity

Two experimental models of hepatotoxicity were used to evaluate the roleof IL-18 and TNF-α in liver injury. Injection of Concanavalin A (Con A)and Pseudomonas aeruginosa (PEA) into mice both induce liver injury, andare models of T cell mediated hepatitis.

C57BL/6J mice were pretreated with an anti-IL-18 antiserum or a solubleTNF-α receptor, TNFsRp55. Serum Alanine aminotransferase (ALT) levelswere measured as indicators of hepatic injury (FIG. 10).

As shown in FIG. 10 A, both IL-18 antiserum and soluble TNF-receptorssignificantly reduced ConA-induced serum ALT levels, as compared to acontrol injection of the vehicle alone (pyrogen free saline). Aco-administration of soluble TNF receptor and IL-18 antiserum led to acomplete inhibition of Con-A induced liver injury.

As shown in FIG. 10 B, in PEA-injected mice, neutralization of eitherTNF-α inhibitors or anti-IL-18 antibodies resulted in 93% and 83%inhibition of serum ALT levels, respectively. A combined blockade ofboth resulted in 99% protection.

Example 23: Plasma Levels of IL-18-Binding Protein are Elevated inPatients with Chronic Liver Disease

IL-18 BP plasma levels were measured in 133 patients with chronic liverdisease (CLD) of varying etiologies and 31 healthy controls by aspecific ELISA, using an IL-18BP monoclonal antibody.

Plasma levels of IL-18 BP were significantly higher in CLD patients(12.91±0.89 ng/ml; average±SEM) than in healthy subjects (4.96±0.43ng/ml, p<0.001). Cirrhotic patients had significantly higher levels thanpatients with non-cirrhotic CLD (19.23±1.28 ng/ml, n=67, vs. 6.49±0.51ng/ml, n=66, p<0.001). Patients with stage B of the Child-Pughclassification had higher levels of IL-18 BP than those with stage A(22.48±2.44 ng/ml vs. 9.57±1.25 ng/ml, p<0.001). However, there was nosignificant difference between Child B and C (22.48±2.44 ng/ml vs.20.62±4.75 ng/ml, p=0.7). Plasma levels of IL-18 BP correlatedpositively with GOT, bilirubin and erythrocyte sedimentation rate.Negative correlation was found with prothrombin time.

In conclusion, the results show that IL-18 BP plasma levels are elevatedin CLD and correlate with the severity of disease independent of theetiology of disease. Although an endogenous antagonist of theproinflammatory IL-18, increased levels of IL-18 BP seem not to besufficient to counteract the overwhelming proinflammatory mediators inCLD.

Example 24: Inhibition of Alcoholic Hepatitis by IL-18BP

Four groups of rats (5 per group) are fed ethanol and a diet containingcorn oil by intragastric infusion for 4 weeks. Dextrose isocaloricallyreplaces ethanol in control rats. The rats are injected daily with mouseIL-18BP (1 mg/kg), or saline. Pathological analysis is performed onliver sections and measurements of liver enzymes in serum, TNF-α, Fasligand and IFN-γ are taken. Necroinflammatory injury and expression ofliver enzymes, TNF-α, Fas ligand, and IFN-γ are seen in the ethanol-fedrats that were injected with saline.

Rats injected with mouse IL-18BP are protected from necroinflammatoryinjury and the levels of liver enzymes, TNF-α, Fas ligand and IFN-γ aresignificantly reduced (>90%).

Example 25: Inhibition of Concanavalin A-induced Hepatitis by IL-18BP

Balb/c mice are injected with 12 mg/kg Concanavalin A (Con A) with orwithout injection of murine IL-18BP (1 mg/kg), 2 h prior to Con Aadministration and then daily. Liver damage is evaluated by determiningserum levels of liver enzymes, TNF-α, Fas ligand and IFN-γ. Hepatichistopathology is compared to mice treated with Concanavalin A only.

Pretreatment with IL-18BP significantly reduces serum levels of liverenzymes and TNF-α with no evidence of inflammation in histopathologicexamination compared to control mice treated with Con A.

PART III: Examples 26 and 27 Relating to the Use of IL-18 Inhibitors inArthritis Example 26: Production of IL-18BP-His Tag

For the experiment described in detail in Example 27 below, recombinanthuman IL-18BP containing a his-tag of 6 residues (r-hIL-18BP-His tag)was produced in CHO cells and purified as described by Kim et al., 2000.The recombinant protein was purified to over 95% purity and found to bebiologically active in-vitro and in-vivo with a high affinity to itsligand.

The production of recombinant proteins in other eukaryotic systems, withor without tags facilitating purification of the recombinant proteins,is known by the person skilled in the art. Well known methods areavailable for constructing appropriate vectors, carrying DNA that codesfor IL-18BP and suitable for transfection of eukaryotic cells in orderto produce recombinant IL-18BP. For expression in cells, the DNA codingfor IL-18BP (see, e.g. Novick et al., 1999) is cut out from the cloningvector and inserted into expression vectors suitable for transfection ofcells. Alternatively, such DNA can be prepared by PCR with suitablesense and antisense primers. The resulting cDNA constructs are theninserted into appropriately constructed eukaryotic expression vectors bytechniques known in the art (Maniatis et al., 1982).

Example 27: Blockade of Endogenous IL-18 in a Murine Model of Arthritis

Methods

Induction of Collagen-Induced Arthritis (CIA)

CIA was induced in male DBA/1 mice (8-12 weeks old) by immunisation withnative type II bovine collagen (CII) as previously described(Plater-Zyberk et al., 1995). From day 25 post-CII immunisation, micewere examined daily for onset of disease.

Treatment with rhIL-18BP-6his

Treatment of CII-immunised DBA/1 mice was started on the firstappearance of clinical sign of disease. Recombinant, human IL-18BPcontaining a tag of 6 histidines (rh-IL-18BPa 6his) was used toneutralise endogenous IL18 in the collagen treated mice. rh-IL-18BP-6hiswas injected daily for 7 seven days at 5 different concentrations 10, 3,1, 0, 5, 0, 25 mg/kg/injection intraperitoneally (i.p.). The placebocontrol mice received the vehicle only (0.9% NaCl).

Assessment of Disease Development

Clinical Evaluation (Clinical Scores)

From first appearance of clinical symptoms, mice were examined every dayby an investigator blinded to the treatment. Each limb was graded fordisease severity (scores 0-3.5, max score=14/mouse). The progression ofedema (inflammation) was measured on the first paws that showed signs ofdisease using precision calipers (Proctest 2T, KroeplinLangenmesstechnik)

Disease progression was assessed daily for 8 days post-onset at whichtime all mice were sacrificed and paws collected for histopathologicalexamination.

Histological Assessment of Cartilage Erosions and MicroscopicalInflammation

At termination of the experiments, i.e. at day 8 post-onset, mice werekilled and the paw that first developed sign of disease was dissectedaway. Joints were fixed, decalcified and embedded in paraffin. Standardsections (5 to 7 μm) of the joints were made and stained withhematoxylin/eosin/Safranin O. Each joint was scored by 2 investigatorsunaware of the treatment protocol (no destruction of cartilage orbone=0; localised cartilage erosions=1-2; more extended erosions=3;general cartilage destruction and presence of bone erosions=4). Thefinal scores of each mouse was the mean of the result on all the scoredjoints. Microsopical inflammation or synovitis was scored from 0 to 4,as follows: no inflammation=0; slight thickening of lining layer and/orsome infiltrating cells in sublining layer=1-2; thickening of lininglayer and/more pronounced influx of cells of sublining layer=3; presencecells in the synovial space and synovium highly infiltrated with manyinflammatory cells=4.

Determination of Anti-Collagen Antibodies.

Antibodies against bovine type II collagen were examined by using anenzyme-linked immunosorbent assay (ELISA). Titers of IgG1 and IgG2a weremeasured. Briefly, plates were coated with 10 μg of bovine collagen andthereafter-nonspecific binding sites were blocked with 0.1 M ethanolamin(Sigma). Serial 1:2 dilutions of the sera were added followed byincubation with isotype specific goat anti-mouse peroxidase (SouthernBiotechnology Associates, Birmingham, Ala., USA) and substrate(5-aminosalicyclic acid, Sigma). Plates were read at 492 nm. Titers wereexpressed as mean±SD dilution, which gives the half-maximal value.

IL-6 Assays

Levels of IL-6 were determined by commercial ELISA (R&D systems,Minneapolis, Minn., USA). IL-6 bioactivity was determined by aproliferative assay using B9 cells. Briefly, 5×10³ B9-cells in 200 μl 5%FCS-RPMI 1640 medium per well were plated in a round-bottom microtitreplate and incubated for 3 days using human recombinant IL-6 (R&Dsystems, Minneapolis, Minn., USA) as standards. At the end of theincubation, 0.5 μCi of ³[H]thymidine (NEN-Dupont, Boston, Mass., USA)was added per well. Three hours later, cells were harvested andthymidine incorporation was determined. Detection limit for the IL-6bioassay was 1 pg/ml.

Statistical Analysis

Significance of differences was assessed by the Mann Whitney test usingSigmaStat statistical analysis program and the GraphPad Prism program.

Results

A mouse experimental model, CIA (collagen induced arthritis), was usedfor assessing the effectiveness of IL-18BP as an agent for the treatmentof arthritis. Administration of collagen and incomplete Freund'sadjuvant in DBA/1 mice induces the development of an erosive,inflammatory arthritis and represents an ideal opportunity to explorethe therapeutic potential of IL-18BP. To this end, endogenous IL-18 wasneutralised using IL-18BP and the effect on various pathogenicparameters was evaluated.

A dose-related study was performed. Three groups of collagen-inducedarthritic DBA/1 mice were treated therapeutically (i.e. after onset ofdisease) with 5 doses of IL-18BP i.p. (intraperitoneal). IL-18BP atconcentrations of 10, 3, 1, 0.5 or 0.25 mg/kg was administered at thefirst clinical sign of disease. Injection of physiological saline(sodium chloride, NaCl) was used as a control. In addition to this, 10000 IU of interferon β (IFN-β) were administered i.p. to assess theeffect of IFN in this experimental model of arthritis. The effect ondisease severity was monitored by daily visual scoring of eachindividual paw as described above. The mice were sacrificed at day 8post-onset.

The following values were measured:

-   -   visual clinical scores (0-3.5 per paw) (FIGS. 11A and B)    -   joint swelling/edema (in mm, measured with calipers) of first        diseased paw, provided it was a hind paw (FIG. 12)    -   number of paws recruited into the disease (FIG. 13)    -   erosion scores of first diseased paw (0-4 cartilage destruction,        FIG. 14).    -   Histopathological analysis of the paw that first developed        arthritis (FIG. 15)    -   Levels of anti-collagen type II antibodies (FIG. 16)    -   Levels of IL-6 (FIG. 17).

Clinical Severity of Disease

As shown in FIGS. 11A and B, the clinical severity of disease wassignificantly diminished in the groups treated with 1 mg/ml (P<0.01) and0.5 mg/ml (0.01<P<0.05) of rhIL-18BP. Mice receiving the low dose ofrhIL-18BP (0.25 mg/kg) or the high dose of 10 mg/kg had a clinical scoresimilar to the placebo group. The dose of 1 mg/kg of IL-18BP wasapproximately as effective as Interferon β (designated IFNb in FIG.11A).

Joint Inflammation and Paw Swelling (Edema)

Macroscopical inflammation (swelling) was studied by measuring paw edemafrom day 1 after onset of disease until day 8, the end of theexperiment. The results are shown in FIGS. 12 A and B. The effectivedoses of IL-18BP were 1, 3 and 10 mg/kg. Administration of lower dosesdid not result in a beneficial effect on the swelling of paws. As shownin FIGS. 12A and 12B, Interferon-β (IFNb) at a concentration of 10 000IU showed a beneficial effect on paw swelling.

Microscopioc synovitis was examined at the end of the experiment onhistopathological sections and was expressed as scores (“synovitisscore”). The results of inflammation (swelling) and synovitis score aresummarized in Table 1. Whilst treatment with rhIL-18BP at dosages 1 and3 mg/kg resulted in a trend towards reduction of swelling, treatmentwith any dosage of IL-18BP had only a limited effect on the inflammatorysynovitis (Table 5).

TABLE 5 EFFECT OF IL-18BP TREATMENT ON JOINT INFLAMMATION Swelling (AUC,Synovitis score Treatment mean ± sem) (mean ± sem) RhIL-18BP-6his 3mg/kg 1.55 ± 0.64 2.17 ± 0.5 1 mg/kg 1.53 ± 0.60 1.98 ± 0.4 0.5 mg/kg3.38 ± 0.70 1.92 ± 0.5 0.25 mg/kg 3.06 ± 0.90 2.08 ± 0.5 Placebo 3.11 ±0.77 2.23 ± 0.3

FIG. 13 shows that the number of paws affected by the disease wasdiminished after administration of IL-18BP. In particular, therapeuticinjections of IL-18BP at doses of 1 and 0.5 mg/kg reduced the number ofpaws recruited into the disease, demonstrating that blockade of IL-18 invivo halts the spreading of arthritis to additional joints. Treatmentwith 1 and 0.5 mg/kg of IL-18BP even appears capable to reverting someof the arthritic joints to normality.

Protection from Joint Destruction

Treatment of mice with rhIL-18BP resulted in protection of joints fromdestruction (FIG. 14). A semi-quantitative scoring system demonstratedthat bone erosion showed a dose-related protective effect that wassignificant at 10 and 3 mg/kg (P<0.05, FIG. 14). Mice receiving 1 mg/kgof rhIL-18BP presented less erosion than mice receiving vehicle only. Noprotection was observed at doses of 0.5 mg/kg and 0.25 mg/kg.Interestingly, the effect on joint protection at doses of 3 and 10 mg/kgIL-18BP were comparable to or even more pronounced than the beneficialeffect of 10 000 IU of Interferon β (IFN-β).

FIG. 15 shows the histology of a healthy (A) and a diseased (B) joint incomparison to a joint derived from an animal treated with IL-18BP (C).Sections were taken at the end of the experiment from those paws whichfirst developed arthritis

The joint from an arthritic mouse showed severe destructive arthritiswith cartilage depletion and erosions and numerous infiltrating cells inthe inflamed synovium. In the joint from a mouse treated with rhIL-18BP,the cartilage appeared almost normal despite the presence ofinflammatory cells in the synovial space. There was not only a higheramount of cartilage, but the cartilage has also a smoother appearance.

Anti-IL-18 Treatment Modulates Levels of Anti-Type II CollagenAntibodies

CIA mice have elevated levels of IgG1 and IgG2a anti-type II collagenantibodies in the circulation. Antibodies of the isotype IgG1 areassociated to TH2 mediated diseases, whereas antibodies of the isotypeIgG2a and IgG2b are associated to TH1 mediated diseases. Arthritis isusually classified as a TH1 mediated disease.

Anti-type II collagen (CII) IgG1 and IgG2a antibody isotypes weredetermined in the sera of animals that were treated with IL-18BP (FIG.16). Levels of anti-CII of the IgG isotypes IgG1 and IgG2a were notsignificantly modified by IL-18BP treatment at day 4 or 8 (D4, D8) ofclinical disease. However, a 2.6 and 3.4 fold decrease incollagen-specific IgG1/IgG2a ratios was observed after 8 days ofrhIL-18BP-treatment, at 1 and 3 mg/kg respectively. FIG. 11 shows theexperiment in which 3 mg/kg were used. Essentially the same results wereobtained using an amount of 1 mg/kg of IL-18BP. The reduced IgG1/IgG2aratio of anti-CII antibodies indicate a diminished concentration ofanti-type II collagen antibodies of the isotype IgG2a and an elevatedconcentration of anti-type II collagen antibodies of the isotype IgG1,suggesting that there is an shift towards TH2-mediated disease in thismodel of arthritis.

Reduction of IL-6 Levels after IL-18 Neutralisation

To gain some insight into the effects of IL-18 blockade, IL-6 wasmeasured in the sera of IL-18BP treated animals. FIG. 17 shows that thelevels of bioactive IL-6 was significantly reduced in the animals havingreceived IL-18BP treatment at all doses measured, i.e. at 1, 3 and 10mg/kg as well as with Interferon-β (IFNb). Immunoactive levels of IL-6measured in the sera of the animals treated with 3 mg/kg rhIL-18BP weresignificantly reduced (P<0.0023) as compared with saline-treatedanimals. IL-6 serum levels of diseased animals treated with 1, 3 or 10mg of IL-18BP or 10 000 IU of IFN-β were similar to normal mouse serum(NMS) derived from healthy animals, i.e. from those animals not havingan inflammatory disease.

These findings demonstrate that IL-18 controls IL-6 levels during theonset of the disease. Since IL-6 is a marker of inflammation, thesefindings show that treatment of diseased mice with IL-18BP reducesinflammation in the animal.

From the experiments outlined above, the following conclusions can bedrawn:

Administration of IL-18BP decreases the clinical severity of arthritis

IL-18BP inhibits further progression or spreading of the disease

Administration of IL-18BP decreases oedema

Administration of IL-18BP decreases cartilage destruction

Serum IL-6 levels are diminished and IgG1/IgG2a anti-CII ratiosdecreased after IL-18BP therapy.

The data presented above indicate that neutralisation of IL-18bioactivity after disease onset represents a disease-modifyinganti-rheumatic therapy. The results clearly demonstrate that IL18blockade reduces the clinical progression of arthritis and moreimportantly stops progression of cartilage and bone destruction. IL18blockade by IL-18BP, anti-IL-18 antibodies or by any other IL-18blocking agent therefore represents a new disease-modifyinganti-rheumatic therapy.

PART IV: Examples 28-34 Relating to the Use of IL-18 Inhibitors inSepsis Example 28: Levels of Serum IL-18BPa and IL-18 in HealthyIndividuals and Sepsis Patients

Measurement of specific circulating cytokines, and their naturalinhibitors in health and disease provides information about theirinvolvement in progression and severity of a disease. As mentioned inthe background section, one of the key mediators of sepsis is IFN-γ.Since IL-18 is a coinducer of IFN-γ, the level of IL-18 and its naturalinhibitor, IL-18BP splice variant a, were monitored in sepsis patientsand compared to the levels found in healthy subjects by using specificELISAs (Example 30).

Levels of IL-18 and IL-18BPa in healthy subjects.

The mean level of IL-18 in 107 healthy donors was 64±17 pg/ml asmeasured by the ECL assay (Pomerantz et al. 2001). The ECL assay wastested for interference by related proteins. It was found that it was noaffected by the presence of mature IL-1β or proIL1β, whereas pro-IL-18was cross-reactive. Therefore, as much as 20% of the detected matureIL-18 in human serum samples in the present study may be pro-IL-18. TheECL assay of IL-18 was not affected by IL18BPa at a concentration ≦160ng/ml.

The levels of IL-18BPa were tested in the serum from the 107 healthyindividuals by ELISA (Example 30). The levels of IL-18BPa ranged from0.5 ng/ml to as high as 7 ng/ml, with an average of 2.15±0.15 ng/ml(FIG. 18).

Because both IL-18 and IL-18BPa are concomitantly present in the serum,some of the IL-18 may be present in a complex with IL-18BPa. The levelof free IL-18 was calculated based on the average level of total IL-18(2.15 ng/ml). Free IL-18 was determined according to the law of massaction. The calculation was based on the following parameters: theconcentrations of total IL-18 as determined by the ECL assay; theconcentration of total IL-18BPa as determined by the ELISA; a 1:1stoichiometry in the complex of IL-18BPa and IL-18 and a dissociationconstant (Kd) of 0.4 nM (Novick et al. 1999 and Kim et al. 2000). In anequilibrium system L+R

LR where L represents IL-18 and R represents IL-18BP the followingequations are applicable:

Kd=[LR]/[L _(free) ][R _(free)]

L _(free) =L _(total) −LR

R _(free) =R _(total) −LR

By substituting: L_(total)=64±17 pg/ml (mean level). R_(total)=2.15±0.15ng/ml (average) and Kd=0.4 nM, it was found that in healthy subjectsabout 51.2 pg/ml IL-18 (about 80% from total) is in its free form.

Levels of IL-18 and IL-18BPa in Sepsis Patients.

The levels of IL-18 and of IL-18BPa were tested in 192 sera samples from42 septic patients immediately upon admission and duringhospitalisation. The levels of both IL-18 and IL-18BPa weresignificantly more elevated in sepsis patients in comparison with thehealthy subjects (FIG. 19A), and a broad distribution of the values wasobserved (FIG. 19 B). Moreover, these levels were even higher in thesepatients at the day of admission, showing a 22 fold increase in thelevel of IL-18 compared with healthy individuals (1.5±0.4 ng/ml versus0.064±0.17 ng/ml) and a 13 fold increase in the level of IL-18BPa(28.6±4.5 versus 2.15±0.15 ng/ml, FIG. 19A). No statisticallysignificant correlation between creatinine levels and either IL-18 orIL-18BPa concentrations in these sera could be observed (assessed byAPACHE II score Knaus et al. 1993), suggesting that elevated IL-18 andIL-18BPa levels in these patients were not due to renal failure.

Because serum IL-18 and IL-18BPa levels in sepsis patients variedconsiderably (FIG. 19B) the levels of free serum IL-18 in individualsamples were calculated. The calculations were done as previouslydescribed, using the same three equations and substitution of the Kd,L_(total), Rtotal values found experimentally. The calculations showthat IL-18BPa reduced the level of free IL-18 in most patients (FIG.20). Notably, this effect was particularly strong when total IL-18 wasvery high. For example, in two patients, serum IL-18 reached 0.5 nM (10ng/ml), a 150-fold increase over healthy individuals. However,considering binding to the circulating IL-18BPa (27 ng/ml and 41.2ng/ml), the level of free IL-18 in these two patients dropped by 78% and84%, respectively (FIG. 20). Therefore, most of the serum IL-18 isblocked by the circulating IL-18BPa. Yet, according to thesecalculations, the remaining free IL-18 is still higher than that foundin healthy individuals. Therefore, administration of exogenous IL-18BPato septic patients is expected to further lower the free circulatingIL-18 level, causing alleviation of the disease outcome.

Example 29: Effect of IL-18BPa on S. epidermidis-induced IFN-γProduction in Cells Present in Whole Blood Samples from Healthy Subjects

The Gram-positive bacteria Staphylococcus epidermidis is known to causesepsis. Induction of cytokines, e.g., IL-1, IFN-γ and TNF-α are keymediators for this pathology. Since IL-18 is a co-inducer of IFN-γ, theeffect of its inhibition on Staphylococcus epidermidis IFN-γ induction,was tested. The IL-18BPa, used as the IL-18 inhibitor, was a recombinanthistidine tagged version produced in CHO cells.

0.5 ml of blood was mixed in 5 ml 12×75 mm round-bottom polypropylenetubes (Falcon, Becton Dickinson Labware, Franklin Lakes, NL) with 0.5 mlof RPMI growth medium (Cellgro Mediatech, Hendon, Va. supplemented with10 mM L-glutamine, 100 U/ml penicillin 100 μg/ml streptomycin and 10%FBS [Gibco BRL, Grand Island, N.Y.]) containing S. epidermidis (fromATCC) with or without IL-18BP. The samples were incubated at 37° C. (5%CO₂) for 48 hours followed by treatment with triton X-100 (Bio-RadLaboratories, Richmond, Calif.) at 0.5% final concentration to lyse thecells present in the whole blood sample. The tubes containing thesamples were inverted several times until the blood sample showed aclear appearance. IFN-γ concentration in the lysed samples of blood,which represents both intracellular and secreted cytokine, was measuredby electrochemiluminescence (ECL) as described by Pomerantz et al.(2001).

For this study whole blood from healthy, non-smoking volunteers wasused. The blood was taken by venipuncture and kept in heparinized tubes.S. epidermidis was propagated in brain-heart infusion broth for 24hours, washed in pyrogen free saline and boiled as described by Aiura etal. (1993). The concentration of heat-killed bacteria was adjusted to 10bacterial cells per white blood cell (WBC).

The results in FIG. 21 show that IL-18BPa is able to inhibit the S.epidermidis induction of IFN-γ.

Since IFN-γ is known to be co-stimulated by IL-18 and IL-12, the effectof the combination of IL-18BPa and anti IL-12 specific antibodies (antihuman IL-12 Mab 11.5.14, Preprotech, Rocky Hill, N.J.) on IFN-γinduction directed by S. epidermidis, was tested. This induction wastested in the presence of 125 ng/ml IL-18BPa (histidine tagged IL-18BPaproduced in COS cells and purified over a talon column as described byNovick et al. 1999) and 2.5 μg/ml anti human IL-12 specific antibody.The results shown in FIG. 22 suggest that anti IL-12 specific antibodiespotentiate the inhibitory effect of IL-18BPa on the IFN-γ induction byS. epidermidis.

Example 30: Development of an IL-18BP Specific ELISA Test

ELISA comprised two anti IL-18BPa antibodies: murine Mab 582.10, asubclone of Mab 582 described in Example 4, as the capture antibody andrabbit polyclonal antibody for detection (Example 4). Microtiter 96-wellELISA plates (Maxisorb; Nunc A/S, Roskilde, Denmark) were coated withanti IL-18BPa MAb No. 582.10 (a subclone of MAb 582, 4 μg/ml in PBS)overnight at 4° C. The plates were washed with PBS containing 0.05%Tween 20 (washing solution) and blocked (2 h, 37° C.) with BSA stocksolution (KPL, Geithersburg, Md.) diluted 1:10 in water. BSA stocksolution was diluted 1:15 in water (diluent) for the dilution of alltested samples and the detecting antibody. Sera samples were diluted atleast 1:5 in the diluent and 100 μl aliquots were added to the wells.Highly pure rIL-18BPa (prepared in CHO and purified by immunoaffinityusing protein G-purified Mab N 430 specific for IL-18BP shown in Table6, example 31) was diluted by 7 serial two-fold dilutions (4 to 0.062ng/ml) and added to each ELISA plate for the generation of a standardcurve. The plates were incubated for 2 hrs at 37° C. and washed 3 timeswith the washing solution. Rabbit anti IL-18BPa serum (1:5000 indiluent, 100 μl/well) was added and the plates were further incubatedfor 2 hrs at 37° C. The plates were washed 3 times, a conjugate ofgoat-anti-rabbit horseradish peroxidase (HRP, Jackson ImmunoResearchLabs, 1:10,000 in PBS, 100 μl/well) was added and the plates wereincubated for 1 h at 37° C. The plates were washed 3 times and developedby the addition of OPD Peroxidase substrate (o-phenylenediaminedihydrochloride tablets, Sigma) for 30 min at room temperature. Thereaction was stopped by 3N HCl (100 μl) and the absorbance at 492 nm wasdetermined by an ELISA reader. The OD was plotted as a function ofIL-18BPa concentration and linearity was observed in a range of0.12-2.00 ng/ml IL-18BPa (FIG. 11).

The IL-18BPa standard curve in the absence of serum or in the presenceof up to 20% human serum remained the same. Since IL-18BPa binds IL-18with a very high affinity, it was necessary to determine whether ELISAdistinguishes between free and bound IL-18BPa. It was found that IL-18interferes with the IL-18 ELISA however, the interference isinsignificant in serum samples. In 90% of sepsis patients, serum IL-18levels were under 4 ng/ml (see below). Such sera samples have elevatedIL-18BPa, requiring a 5 to 20-fold dilution prior to measuring IL-18BPa.At such dilutions, IL-18 interference with the IL-18BPa ELISA is lessthan 10% (FIG. 24). Other related cytokines, including proIL-18, used ata 10-fold molar excess over IL-18BPa, as well as a 200-fold excess ofmature IL-1β do not interfere with the assay.

The most abundant isoform of human IL-18BP is IL-18BPa, whereas isoformsb, c and d are minor splice variants (Novick et al. 1999 and Kim et al.2000). IL-18BPa exhibits the highest affinity for IL-18 (Kim et al2000). The affinity of IL-18BPc for IL-18 is 10 fold lower, whereasisoforms b and d do not bind or neutralize IL-18. It was thereforeimportant to determine the cross-reactivity of IL-18BPa in ELISA withthe other isoforms of IL-18BP. As shown in FIG. 25, human IL-18BPcgenerated a 10-fold lower signal on a weight basis compared withhuIL-18BPa, whereas human isoforms b and d generated an insignificantsignal in the ELISA.

Example 31: Generation of Anti IL-18BP Specific Antibodies

A rabbit was injected with rIL-18BPa-His6 for the generation ofpolyclonal antibodies.

For the production of monoclonal antibodies, female Balb/C mice wereinjected 5 times with 10 μg of recombinant histidine tagged IL-18BPa(rIL-18BPa-His6). The mouse exhibiting the highest titer as determinedby an inverted radioimmunoassay (IRIA example 32) or solid phase RIA(sRIA, example 32) was given a final boost intraperitoneally 4 and 3days before fusion. Lymphocytes were prepared from spleen and fusion toNSW myeloma cells was performed. Hybridomas that were found to produceantibodies to IL-18BPa were subcloned by limiting dilution. The clonesgenerated were assayed by IRIA (Example 32). Representative hybridomasare shown in Table 6.

TABLE 6 Representative hybridomas producing anti IL-18BPa MAb No. IRIA₁(cpm) sRIA₂ (cpm) 148 23912 7941 297 1652 6483 369 316 12762 430 68873254 433 1009 15300 460 3199 4326 485 400 13010 582 15000 17897 601 10461928 ₁Plates coated with goat anti mouse antibodies and hybridomasdetected with 1251 IL 18BPa. ₂Plates coated with urinary IL 18BP andhybridomas detected with 125I goat anti mouse antibodies.

Hybridomas secreting antibodies directed against the histidine tag werediscarded. Antibodies were further characterized by sRIA for theirability to recognize the naturally occurring IL-18BP (purified fromurine). Binding characteristics of several positive hybridomas are shownin Table 3. Hybridomas No. 148, 430, 460 and 582 were positive withboth, recombinant and urinary IL-18BP. Antibodies suitable for Westernblotting, immunoprecipitation, immunoaffinity purification and fordevelopment of a specific ELISA were obtained. Positive clones producinganti IL-18BP specific antibodies were injected into Balb/C micepre-primed with pristane for the production of ascites. The isotypes ofthe antibodies were defined with the use of an anti mouse IgG ELISA(Amersham-Pharmacia Biotech). Mab 582, which was highly reactive withthe recombinant and the native IL-18BP (Table 6) was used for theassembly of the IL-18BP specific ELISA (Example 30).

Antibodies were tested by sRIA in the presence of IL-18 (Example 32) fortheir ability to recognize a complex of IL-18BPa with IL-18. Mostantibodies were unable to recognize IL-18BP when it was complexed toIL-18. Therefore, these antibodies appear to be directed against theligand-binding domain of IL-18BPa.

Example 32: Radioimmunoassays for the Detection of Anti IL-18BP AntibodyProducing Cell Clones

Inverted Radioimmunoassay (IRIA).

PVC microtiter plates (Dynatech Laboratories, Alexandria, Va.) werecoated overnight at 4° C. with affinity-purified goat anti-mouse F (ab)2 antibodies (10 μg/ml, 100 μl/well; Jackson ImmunoResearch Labs). Theplates were then washed twice with PBS containing 0.05% Tween 20(washing solution) and blocked with BSA (0.5% in washing solution) for 2hrs at 37° C. Hybridoma culture supernatants (100 μl/well) were addedand the plates were incubated for 2 hrs at room temperature. The plateswere washed three times, ¹²⁵I-rIL-18BPa-His6 (105 cpm in 100 μl) wasadded to each well and the plates were incubated for 5 hrs at 22° C. Theplates were then washed three times and individual wells were counted ina gamma counter. Hybridomas generating supernatants, which exhibitedbound radioactivity at levels 5 folds higher than the negative control,were considered positive.

Solid Phase RIA (sRIA).

rIL-18BPa (5 μg/ml) was used as the capture antigen and ¹²⁵I-goat antimouse antibodies (100 μl, 10⁵ cpm) were used for detection. Blocking andwashings were done as above. Positive clones were further screened bysRIA for their ability to recognize the IL-18BP isolated fromconcentrated human urine (Novick et al. 1999). Microtiter plates werecoated with ligand-affinity purified urinary IL-18BP (1 μg/ml), blockedand washed as above. Hybridoma supernatants (100 μl) were added anddetection was done with ¹²⁵I-goat anti mouse antibodies (10⁵ cpm in 100μl).

sRIA for Antibodies Specific for the Ligand-binding Site of IL-18BP.

Microtiter plates were coated with either urinary IL-18BP (0.5 μg/ml) orrIL-18BPa (5 μg/ml), blocked and washed as in sRIA. Recombinant humanIL-18 (50 μl) was added to a final concentration of 1.5 μg/ml (15 min atroom temperature), followed by the addition of hybridoma supernatants(50 μl). Detection was done with ¹²⁵I-goat anti mouse antibodies (10⁵cpm in 100 μl, 2 h at room temperature).

Example 33: Myocardial IL-18 Content Following LPS

TNFα and IL-1β have been implicated in cardiac dysfunction duringsepsis, since IL-18 is a pro-inflammatory cytokine known to mediate theproduction of TNFα and IL-1β, the concentration of IL-18 on LPS-inducedcardiac dysfunction was tested.

Mice were treated with either vehicle (saline) or LPS. Hearts wereharvested at 2, 4 and 6 hours following LPS administration andhomogenized to determine myocardial IL-18 content by ELISA (Kitpurchased from R&D Systems (Minneapolis Minn.). The results in FIG. 26show a two-fold increase in myocardial IL-18 content at 4 hoursfollowing LPS administration, which indicates that IL-18 is involved incardiac dysfunction during sepsis.

Example 34: Effect of Neutralization of IL-18 on LPS-Induced MyocardialDysfunction

In the previous example an increase in the IL-18 content has been foundin cardiac dysfunction induced by LPS. Therefore, the effect ofneutralization of IL-18 on LPS-induced myocardial dysfunction wasevaluated.

Myocardial function was determined by an isovolumetric, nonrecirculatingLangendorff technique as described previously (Meng et al. 1998).Isolated hearts were perfused with normothermic Krebs-Henseleit solutioncontaining 11.0 mmol/l glucose, 1.2 mmol/l CaCl₂, 4.7 mmol/l KCL, 25mmol/l NaHCO₃, 119 mmol/l NaCl, 1.17 mmol/l MgSO₄ and 1.18 mmol/lKH₂PO₄. A latex balloon was inserted in the left ventricle via the leftatrium and inflated with water to achieve a left ventricular anddiastolic pressure (LVEDP) of 10 mmHg. Pacing wires were attached to theright atrium and hearts were paced at 300 beats per minute. Coronaryflow was quantified by collecting the effluent from the pulmonaryarteries. Myocardial temperature was maintained at 37° C. Leftventricular developed pressure (LVDP), its first derivatives (+dP/dt,−dP/dt) and LVEDP were continuously recorded by a computerized pressureamplifier-digitizer (Maclab 8, AD Instrument, Cupertino, Calif.). Aftera 20 minutes equilibration period LVDP and +/−dP/dt were determined atvaried LVEDP levels (10, 15, and 20 mmHg).

Following LPS administration, the left ventricular developed pressure(LVDP) was reduced by 38% compared to saline controls (36.3+/−1.9 mmHgvs. 59.1+/− mmHg, P<0.001, FIG. 27). Pre treatment of mice 30 minutesprior to LPS administration with normal rabbit serum (NRS) had minimalinfluence on LPS-induced myocardial dysfunction; however pretreatmentwith IL-18 neutralizing antibody abrogated dysfunction (FIG. 27).Coronary flow was not different between the groups (not shown).

The results indicate that neutralization of IL-18 protects againstLPS-induced myocardial dysfunction.

The foregoing description of the specific embodiments reveal the generalnature of the invention so that others can, by applying currentknowledge, readily modify and/or adapt for various applications suchspecific embodiments without departing from the generic concept, and,therefore, such adaptations and modifications should and are intended tobe comprehended within the meaning and range of equivalents of thedisclosed embodiments. It is to be understood that the phraseology orterminology employed herein is for the purpose of description and not oflimitation.

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1. A method for treatment and/or prevention of a sepsis and otherdiseases characteristic to the Systemic Inflammatory Response Syndrome(SIRS) comprising administering to a subject in need thereof apharmaceutically effective amount of an inhibitor of IL-18 selected froma group consisting of caspase-1 (ICE) inhibitors, antibodies againstIL-18, antibodies against any of the IL-18 receptor subunits, inhibitorsof the IL-18 signaling pathway, antagonists of IL-18 which compete withIL-18 and block the IL-18 receptor, and inhibitors of IL-18 production,and a pharmaceutically acceptable carrier.
 2. The method according toclaim 1, further comprising administering a therapeutically effectiveamount of an IL-12 inhibitor.
 3. The method according to claim 1,further comprising administering a cytokine inhibitor selected from agroup consisting of a Tumor Necrosis Factor (TNF) inhibitor, an IL-1inhibitor and an IL-8 inhibitor.
 4. The method according to claim 2,further comprising administering a cytokine inhibitor selected from agroup consisting of a Tumor Necrosis Factor (TNF) inhibitor, an IL-1inhibitor and an IL-8 inhibitor.
 5. The method according to claim 3,wherein the TNF inhibitor is a soluble portion of TNFRI or TNFRII. 6.The method according to claim 1, wherein the method further comprisesadministering an interferon.
 7. The method according to claim 2, whereinthe method further comprises administering an interferon.
 8. The methodaccording to claim 3, wherein the method further comprises administeringan interferon.
 9. A method according to claim 6, wherein the interferonis interferon-α or interferon-β.
 10. A method for the treatment and/orprevention of sepsis and other diseases characteristic to the SystemicInflammatory Response Syndrome (SIRS) comprising administering to asubject in need thereof a pharmaceutically effective amount of a vectorcoding the sequence of an inhibitor of IL-18 selected from a groupconsisting of caspase-1 (ICE) inhibitors, antibodies against IL-18,antibodies against any of the IL-18 receptor subunits, inhibitors of theIL-18 signaling pathway, antagonists of IL-18 which compete with IL-18and block the IL-18 receptor, inhibitors of IL-18 production, and IL-18binding proteins, isoforms, muteins, fused proteins or circularlypermutated derivatives thereof and a pharmaceutically acceptablecarrier.
 11. A method for the treatment and/or prevention of sepsis andother diseases characteristic to the Systemic Inflammatory ResponseSyndrome (SIRS) comprising administering to a subject in need thereof apharmaceutically effective amount of i) a vector for inducing and/orenhancing the endogenous production of an inhibitor of IL-18 in a cellor ii) a cell that has been genetically modified to produce an inhibitorof IL-18.
 12. The method according to claim 1, wherein the sepsis isrelated to cardiac dysfunction.
 13. The method according to claim 10,wherein the fused protein comprises an Ig fusion.
 14. The methodaccording to claim 1, wherein the inhibitor of IL-18 is an anti IL-18specific antibody selected from chimeric, humanized and humanantibodies.
 15. The method according to claim 2, wherein the IL-12inhibitor is a neutralizing antibody.